Functional heavy chain antibodies, fragments thereof, library thereof and methods of production thereof

ABSTRACT

The present invention relates to functional heavy chain antibodies, functional single domain heavy chain antibodies, functional VH domains, or functional fragments thereof comprising an amino acid which is neither a charged amino acid nor a C at position 45, and comprising an amino acid at position 103 independently chosen from the group consisting of R, G, K, S, Q, L, and P, and optionally an amino acid at position 108 independently chosen from the group consisting of Q, L and R, said positions determined according to the Kabat numbering.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/474,521, filed Jun. 23, 2006, which is a continuation of U.S.application Ser. No. 10/492,668, filed Oct. 5, 2004, which is nationalstage filing under 35 U.S.C. §371 of international applicationPCT/EP02/07804, filed Jul. 12, 2002, which was published under PCTArticle 21(2) in English, and which claims the benefit under 35 U.S.C.§119(e) of U.S. provisional application Ser. No. 60/355,054, filed Oct.24, 2001, all of which are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to functional heavy chain antibodies,fragments thereof and a library thereof. It also relates to methods forproducing functional heavy chain antibodies, fragments thereof and alibrary thereof. It further relates to uses of functional heavy chainantibodies, fragments thereof and a library thereof.

BACKGROUND OF THE INVENTION

The IgG isotype is the most abundant immunoglobulin found in sera. Inall mammals, it is composed of two identical heavy (H) chains and twoidentical light (L) chains. Immunoglobulins harbouring this structureare therefore designated four-chain immunoglobulins. The H-chain of a4-chain immunoglobulin contains 4 domains and a hinge region in betweenthe second and third domain. The L-chain has two domains. The N-terminaldomains of both the L- and H-chain are more variable in sequence thanthe remaining domains and are known as V-domains (VL and VHrespectively). Three loops within the VH and three loops within the VLjuxtapose in the paired VH-VL domains and constitute the antigen-bindingsite. The loops are hypervariable in sequence and named CDR forComplementarity Determining Region. A description of the generalstructure of a 4-chain Immunoglobulin is provided in “Immunology” RoittI. et al., Ed. MEDSI/McGRAW-HILL.

Much of the antigen binding diversity and the success of antibodies togenerate a tight antigen binder against virtually all possible foreignsubstances, comes from the random pairing of one out of thousands ofpossible VHs with one out of thousands possible VLs. The second domainof the L-chain, having a more conserved sequence and denoted CL, isassociated with the second domain of the H-chain (CH1) that has also aconserved sequence.

A pathological disorder in humans, known as heavy chain disease, ischaracterised by the presence of antibodies in the serum that do notcontain L-chains. Moreover, these antibodies lack important parts oftheir VH and CH1 as well, although the missing VH and CH1 regions canvary widely among different HCAb (Heavy Chain Antibody). The deletionsin the H-chain are due to deletions of the rearranged H-chain involvingpart of the VH and the CH1 domain. These antibodies no longer recogniseantigen since the VL is absent and large parts of the VH is absent too.The HCAb can be secreted from the B-cells because the chaperone proteins(such as BIP) that associate with the CH1 retain the H chain in theendoplasmic reticulum until BIP is replaced by the L-chain. In absenceof the CH1 polypeptide domain, the BIP can no longer retain thetruncated H-chain in the endoplasmic reticulum, and the L-chain cannotbind either resulting in the fact that the H-chains are immediatelysecreted as homodimers.

Similar non-functional HCAbs were also reported to emerge in mousemonoclonal cell lines.

In sera of Camelidae (camels, dromedaries and llamas) we found thepresence of the 4-chain immunoglobins and, in addition, of large amountsof functional HCAbs. These functional HCAbs have been described inEuropean Patent Application No. 0656946 and in various publicationsincluding Hamers-Casterman et al. (1993), Vu et al. (1997) andMuyldermans et al. (2001). They are distinct from the human/mouse HCAbspresent as a result of the pathological stage, in several respects.Firstly, they are functional in antigen binding. In this respect theHCAbs found in Camelidae are functional normal immunoglobulins.Secondly, in Camelidae, the entire CH1 domain is missing, and the Vdomain is intact but HCAbs have a sequence that deviates at a few sitesfrom normal VH sequences. Said functional HCAb occur as a homodimericmolecules.

The CH1 is however encoded in the germline of all γ-genes in dromedaries(and llama) and is removed from the mRNA coding for the functional HCAbsby a splicing of the 3′ end of the V-exon with the 5′ end of the hingeexon. Thus, the CH1 is part of the intron and is no longer recognized asan exon because of a single point mutation of the consensus splicingsignal sequence. Llama and dromedary carry the same point mutation atthe former CH1 exon and this finding indicates that these γ-genesemerged before the llama and camels diverged from each other. Thedifferent splicing activity of the mRNA is not an alternative splicingas all mRNA is spliced according to this scheme. Hence these γ-geneswill always lead to a H-chain with its CH1 removed. Other γ-genes areused to produce the common H-chain with a CH1 domain.

The V-domain of the H-chain of functional HCAb (referred to as VHH, forVariable domain of the H-chain of a normal, i.e. immunologically-activeHCAb) is expected to acquire adaptations versus the VH (i.e. V-domain ofH-chain of conventional four-chain antibody) in the regions that are nolonger contacting the VL (or the CH1) domain and in those participatingin antigen binding (i.e. the paratope).

For instance, Chothia et al. (1985) have indicated in theabove-referenced publication that crystallographic data revealed thatconserved Val 37, Gly 44, Leu 45 and Trp 47 are clustered in space in aconventional 4-chain IgG and make important hydrophobic contacts withthe VL. They added that the VH amino acids Gln 39, Tyr 91, Trp 103 andGlu 105 are also recognized as important for VL association. Desmyter etal. (1996) further observed that the surface of the VHH domain which ispresent in camelidae and which corresponds to the VH side ofconventional IgG which interacts with a VL is significantly reshaped inthe camelid VHHs. In the present invention, the numbering of the aminoacid residues is given by reference to the Kabat numbering (Kabat E,1991) which is used in accordance with the Kabat database available, forexample at the website of Dr. A.C.R. Martin's Bioinformatics Group atUniversity College London bioinf.org.uk/abs.

The most frequently occurring amino acid residues at twelve VH locationsknown to interact with VL have been determined for 332 vertebrate VHsegments. It is mentioned that for the purpose of the present invention,the protein domain of the variable heavy polypeptide chain is referredas “VH” and the corresponding DNA is designated VH-D-J as it isassembled from a VH germline, a diversity D minigene and a J minigene.In fact the CDR3 and FR4 are not encoded by the VH, but they areprovided by D and J minigene that are recombined with the VH or (VHH)germline.

For comparison, the amino acid consensus has been deduced for 42dromedary germline VHH sequences at the corresponding locations. Thepreferred amino acid residues at four positions (39, 43, 60 and 91,Kabat numbering) is invariable in VH and VHH. In contrast, at four othersites (33, 35, 50 and 58) neither VH nor VHH sequences reveal apronounced amino acid preference. At the latter VH sites, the possiblecontact with the VL is dependent on the actual angle between VH and VLdomains, and this explains the observed amino acid degeneracy. The onlycrucial differences between VH and VHH proteins in this area concernposition 37, 44, 45 and 47. These are highly conserved amino acidresidues among VH phenotypes (i.e. Val37, Gly44, Leu45 and Trp47), butin the VHH, the inventors observed most frequently Phe37 (or Tyr),Glu44, Arg45 (or Cys), and Gly47 (or Leu). These comparisonssubstantiate previous identifications of camel VHH-specific “hallmark”residues that arise in response to the absence of the L-chain.

From the results published by Nguyen et al. (2000), it is apparent thatVHH and VH genes are imprinted in the dromedary genome. The VH and VHHgenes are most likely residing in the same locus. It was noticed thatthe VH and VHH germline genes use the same D and Jgenes with the H-chainof conventional 4-chain antibodies. By PCR, around 50 VH and around 40VHH germline genes were identified in dromedary. Each PCR fragmentcontains a leader signal exon and a V-exon, that ends where the CDR3should start. The CDR3 and FR4 are provided by the recombined D-Jsegments. The VH germline segment harbours codons for Val37, Gly44,Leu45 and Trp47, and the VHH germline minigenes possess the Phe37 (11×)Tyr37 (30×) or in one single case Val37; Glu44 or Gln44 (8×); Arg45(37×) or Cys45 (5×) and Gly47 (6×) or Leu47 (24×) or Trp47 (8×) or Phe47(3×). In addition, these VHH germline-genes contain always (except 1) aCys codon at position 45 or at the CDR1 region (codon 30, 32 or 33).Based on the length of the CDR2 (16 or 17 amino acids in size) and thelocation of the extra Cys, the VHH germline segments were grouped insubfamilies. Some subfamilies had several members while others are muchscarcer in the genome. However, it should be noted that the frequency ofoccurrence of these VHH germline genes in expressed HCAb is not at allrelated to their frequency of occurrence in the genome. The Cys atposition 45 or around the CDR1 is normally maintained in the rearrangedVHH-D-J segments, and these rearrangements products have also acquiredan extra Cys in the CDR3. Likewise, VHH-D-J rearrangements that wereunable to generate an extra Cys in their CDR3 will apparently knock outthe Cys45 or Cys in the CDR1 region probably by somatic hypermutation orby B-cell receptor editing. B cell receptor editing is a mechanism bywhich an upstream unrearranged V-segment is recombined into an existingV-D-J recombination product, that was most likely not functional, orrecognizing a self antigen.

For dromedary, the VHH domains carry also longer CDR3 than that of theVH domains (average length 17-18 versus 9). Three possibilities can beenvisaged to generate a longer CDR3. The VHH may uses two or more Dminigenes, however, this is unlikely in view of the necessity torecombine two minigenes with a different recombination signal sequence(the 12-23 spacer rule). Alternatively, a more active terminaldeoxynucleotidyl transferase during the D-J or V-D-J recombination mightadd several non-template encoded nucleotides. Finally, it can not beexcluded that the length difference is only due to selection in whichthe fraction of VHH domains with long CDR3 or the VH domains with shortCDR3 is much more likely to become functional to interact with antigen.A combination of the two latter explanations might also be relevant.

It has been proposed repeatedly that the presence of the VHH hallmarksat positions 37, 44, 45 or 47 or the substitution of the VH into the VHHhallmarks can lead to the formation of soluble single-domain antibodyfragment. Of these, the amino acid at position 45 was considered crucialas the substitution of Leu45 of a human VH domain by Arg45 rendered theisolated domain more soluble. This camelised human VH adopts a properlyfolded immunoglobulin structure (Riechman, 1996. Rearrangement of theformer VL interface in the solution structure of a camelised, singledomain VH antibody).

However, work of Chothia et al. (1985) revealed that amino acids of VHat position 35, 37, 39, 44, 45, 47, 91, 93 encoded by the VH genesegment, 95, 100, 101 as part of the CDR3, and 103, 105 encoded by the Jgene segment are the key participants for the VL interaction. Of these,amino acids 37, 45, 47 differ largely between VH and VHH. Position 103is occupied by a conserved Trp that is well buried in the VH-VL complexand provides the largest contact surface area with the VL after Leu45and Trp47 (FIG. 2 in Chothia et al.). As this Trp103 is encoded by the Jgene and as the J gene is used in common in the VH-D-J and VHH-D-Jrecombination, it is logical to expect Trp at position 103 in VHH's aswell. Since the VH-VL association is mediated by hydrophobicinteractions, it is also clear that the substitution of the largearomatic and hydrophobic Trp 103 residue by the charged and hydrophilicArg will prevent the association with a VL, and that of the surrogatelight chain as well. WO92/01787 claims a single chain variable domain,being a synthetic variable immunoglobulin heavy chain domain, in whichone or more of the amino acid residues at position 37, 39, 45, 47, 91,93 or 103 is altered, whereby the tryptophan at position 103 is changedinto glutamate, tyrosine or threonine. However, there is no indicationthat a substitution of tryptophan at 103 alone by arginine, glycine,lysine, proline or serine would be sufficient to obtain a functionalheavy chain antibody, neither that this mutation could compensate forthe absence of a charged amino acid or a cysteine at position 45, northat said mutation may result in an increased solubility of a singledomain heavy chain antibody fragment.

It is known in the art that the production of antibodies, for example bybacterial overexpression techniques, by phage display libraries, istechnically difficult due to the antibody or fragments thereof beingpoorly expressed, insoluble, mis-folded. It is also known that thescreening of antibody libraries is restricted to those which aresoluble, so excluding a large fraction of antibodies with potentiallyactive antigen binding regions. Thus binders which might betherapeutically useful would be precluded from screening. There is aneed by researchers involved in discovering new therapeutic agents for amethod for producing functional antibodies and fragments thereof. Thereis a need by researchers involved in discovering new therapeutic agentsfor antibody libraries comprising functional antibodies. There is a needby researchers involved in discovering new therapeutic agents formethods to functionalise antibodies.

SUMMARY OF THE INVENTION

One embodiment of the invention is a functional heavy chain antibody, afunctional single domain heavy chain antibody, a functional VH domain,or a functional fragment thereof comprising, an amino acid which isneither a charged amino acid nor a C at position 45, and comprising anamino acid at position 103 independently chosen from the groupconsisting of R, G, K, S, Q, L, and P, and optionally an amino acid atposition 108 independently chosen from the group consisting of Q, L andR, said positions determined according to the Kabat numbering.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof comprising an amino acidwhich is neither a charged amino acid nor a C at position 45 and SEQ IDNO: 13 (RGQGTQ) according to FIG. 6, said positions determined accordingto the Kabat numbering.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof comprising an amino acidwhich is neither a charged amino acid nor a C at position 45 and SEQ IDNO: 14 (RGKGTQ) according to FIG. 6, said positions determined accordingto the Kabat numbering.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof comprising SEQ ID NO: 15(VXXXXXXGLXW) according to FIG. 6, wherein X is any amino acid, saidpositions determined according to the Kabat numbering.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof comprising an amino acidwhich is neither a charged amino acid nor a C at position 45 and SEQ IDNO: 16 (LGQGTQVTVSS) according to FIG. 6, said positions determinedaccording to the Kabat numbering.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof comprising an amino acidwhich is neither a charged amino acid nor a C at position 45 and SEQ IDNO: 17 (QGQGTGVTVSS) according to FIG. 6, said positions determinedaccording to the Kabat numbering.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof comprising an amino acidwhich is neither a charged amino acid nor a C at position 45 and SEQ IDNO: 18 (PGQGTQVTVSS) according to FIG. 6, said positions determinedaccording to the Kabat numbering.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof comprising an amino acidwhich is neither a charged amino acid nor a C at position 45 and SEQ IDNO: 19 (SSQGTQVTVSS) according to FIG. 6, said positions determinedaccording to the Kabat numbering.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising any one of SEQID NOS: 1 to 10 according to FIG. 6.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising any one of SEQID NOS: 1, 3, 5, 7 or 9 according to FIG. 6.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising any one of SEQID NOS: 2, 4, 6, 8 or 10 according to FIG. 6.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising any one of SEQID NOS: 1, 2, 3, 4 or 5 according to FIG. 6.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising any one of SEQID NOS: 6, 7, 8, 9 or 10 according to FIG. 6.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising any one of SEQID NOS: 5, 6, 7, 8 or 9 according to FIG. 6.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising any one of SEQID NOS: 1, 3, 7, 9 or 10 according to FIG. 6.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising any one of SEQID NOS: 2, 5, 8, 9 or 10 according to FIG. 6.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising any one of SEQID NOS: 3, 4, 5, 6 or 7 according to FIG. 6.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising any one of SEQID NOS: 4, 6, 7, 8 or 9 according to FIG. 6.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising any one of SEQID NOS: 20 to 79 according to FIG. 6.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 1.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 2.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 3.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 4.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 5.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 6.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 7.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 8.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 9.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 10.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 20.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 21.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 22.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 23.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 24.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 25.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 26.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 27.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 28.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 29.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 30.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 31.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 32.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 33.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 34.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 35.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 36.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 37.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 38.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 39.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 40.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 41.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 42.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 43.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 44.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 45.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 46.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 47.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 48.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 49.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 50.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 51.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 52.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 53.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 54.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 55.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 56.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 57.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 58.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 59.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 60.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 61.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 62.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 63.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 64.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 65.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 66.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 67.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 68.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 69.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 70.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 71.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 72.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 73.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 74.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 75.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 76.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 77.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 78.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, comprising SEQ ID NO: 79.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, according to any of themacromolecules above wherein said macromolecule is derived from camel.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, according to any of themacromolecules above wherein said macromolecule is derived from human.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, according to any of themacromolecules above wherein said macromolecule is derived from mouse.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, according to any of themacromolecules above wherein said macromolecule is derived from rabbit.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, according to any of themacromolecules above wherein said macromolecule is derived from goat.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, according to any of themacromolecules above wherein said macromolecule is derived fromkangaroo.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, according to any of themacromolecules above wherein said macromolecule is derived from sheep.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, according to any of themacromolecules above wherein said macromolecule is derived from anyvertebrate species other than camel, human and mouse.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof according to any of themacromolecules above, as an artificial mutant.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, according to any of themacromolecules above, as a peptide homologue of said functional heavychain antibody, functional single domain heavy chain antibody,functional VH domain, or functional fragment thereof.

By “homologue” as meant herein is an amino acid sequence which is atleast 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%,99.5% identical to the amino acid sequences of the present invention. Bya polypeptide with an amino acid sequence having at least, for example,95% “identity” to a reference amino acid sequence of the presentinvention, it is intended at the amino acid sequence of the polypeptideis identical to the reference sequence, except that it may have up to 5%of its amino acids deleted or substituted compared with the referencesequence, or, except that the sequence may have amino acid insertions upto 5% of the total number of amino acids in the reference sequence. As apractical matter, whether any particular peptide is at least 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% identical tothe amino acid sequences of the present invention can be determinedusing known algorithms.

Another embodiment of the invention is a polypeptide comprising afunctional single domain heavy chain antibody, a functional VH domain,or a functional fragment thereof, as described above.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, as described above whereinone or more amino acids are derivatized.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof as defined above whereinsaid macromolecules are dimers.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof as defined above whereinsaid macromolecules are trimers.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof as defined above whereinsaid macromolecules are multimeric.

Another embodiment of the invention is a method to functionalize a heavychain antibody, a single domain heavy chain antibody, a VH domain, or afragment thereof by replacing the amino acid at position 103 with anamino acid independently chosen from the group consisting of R, G, K, Sand P, and optionally replacing the amino acid at position 108 with anamino acid independently chosen from the group consisting of L, Q and R,said positions determined by the Kabat numbering.

Another embodiment of the invention is a method to functionalize a heavychain antibody, a single domain heavy chain antibody, a VH domain, or afragment thereof by replacing the amino acid at position 103 with R,said position determined according to the Kabat numbering.

Another embodiment of the invention is a method to humanize andfunctionalize a heavy chain antibody, a single domain heavy chainantibody, a VH domain, or a fragment thereof, said method comprisingreplacing the amino acid at position 45 with L, and optionally replacingthe amino acid at position 37 with V and/or the amino acid at position44 with G and/or the amino acid at position 47 with W, and replacing ofamino acid at position 103 with R, said position determined according tothe Kabat numbering.

Another embodiment of the invention is a method to humanize andfunctionalize a heavy chain antibody, a single domain heavy chainantibody, a VH domain, or a fragment thereof, said method comprisingreplacing the amino acid at position 45 with L, replacing the amino acidat position 103 with an amino acid independently chosen from the groupconsisting of R, G, K, S and P, and optionally replacing the amino acidat position 37 with V and/or the amino acid at position 44 with G and/orthe amino acid at position 47 with W, and optionally replacing the aminoacid at position 108 with an amino acid independently chosen from thegroup consisting of L, Q and R, said positions determined by the Kabatnumbering.

Another embodiment of the invention is a method according to the abovemethods, wherein said heavy chain antibody, single domain heavy chainantibody, VH domain, or fragment thereof is derived from human or mouse.

Another embodiment of the invention is a method according to the abovemethods, wherein said heavy chain antibody, single domain heavy chainantibody, VH domain, or fragment thereof is derived from human.

Another embodiment of the invention is a method according to the abovemethods, wherein said heavy chain antibody, single domain heavy chainantibody, VH domain, or fragment thereof is derived from mouse.

Another embodiment of the invention is a method according to the abovemethods, wherein said heavy chain antibody, single domain heavy chainantibody, VH domain, or fragment thereof is derived from rabbit.

Another embodiment of the invention is a method according to the abovemethods, wherein said heavy chain antibody, single domain heavy chainantibody, VH domain, or fragment thereof is derived from goat.

Another embodiment of the invention is a method according to the abovemethods, wherein said heavy chain antibody, single domain heavy chainantibody, VH domain, or fragment thereof is derived from sheep.

Another embodiment of the invention is a method according to the abovemethods, wherein said heavy chain antibody, single domain heavy chainantibody, VH domain, or fragment thereof is derived from rat.

Another embodiment of the invention is a method according to the abovemethods, wherein said heavy chain antibody, single domain heavy chainantibody, VH domain, or fragment thereof is derived from any vertebratespecies other than human and mouse.

Another embodiment of the invention is a method to humanize a functionalcamelid heavy chain antibody, a functional camelid single domain heavychain, a functional camelid VHH domain or a functional fragment thereof,said method comprising replacing the amino acid at position 45 with L,and optionally replacing the amino acid at position 37 with V and/or theamino acid at position 44 with G and/or the amino acid at position 47with W, said positions determined by the Kabat numbering.

Another embodiment of the invention is a method to camelize a functionalheavy chain antibody, a functional single domain heavy chain, afunctional VH domain or a functional fragment thereof, said methodcomprising replacing the amino acid at position 45 with an amino acidindependently chosen from the group consisting of L, V and P, replacingthe amino acid at position 103 with an amino acid independently chosenfrom the group consisting of R, G, K, S and P, and replacing the aminoacid at position 37 with F, the amino acid at position 44 with G, theamino acid at position 47 with W, and amino acid at position 103 with R,and optionally replacing the amino acid at position 108 with an aminoacid independently chosen from the group consisting of L, Q and R, saidpositions determined by the Kabat numbering.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, obtainable by the methodsabove.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, obtained by the methodsabove.

Another embodiment of the invention is a functional humanized camelidheavy chain antibody, a functional humanized camelid single domain heavychain, a functional humanized VH domain or a functional fragmentthereof, obtainable by the methods above.

Another embodiment of the invention is a functional humanized camelidheavy chain antibody, a functional humanized camelid single domain heavychain, a functional humanized VH domain or a functional humanizedfragment thereof, obtained by the method above.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody; a functionalVH domain, or a functional fragment thereof, according to above 4paragraphs, as an artificial mutant.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, according to above 5paragraphs, as a peptide homologue of said functional heavy chainantibody, functional single domain heavy chain antibody, functional VHdomain, or functional fragment thereof.

Another embodiment of the invention is a polypeptide comprising afunctional heavy chain antibody, a functional single domain heavy chainantibody, a functional VH domain, or a functional fragment thereofaccording to according to above 6 paragraphs.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof, according to the above 7paragraphs which recite said macromolecules, or a polypeptide accordingthe above paragraph wherein one or more amino acids is derivatized.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof obtained by the methods asdefined above wherein said macromolecules are dimers.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof obtainable by the methods asdefined above wherein said macromolecules are dimers.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof obtained by the methods asdefined above wherein said macromolecules are trimers.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof obtainable by the methods asdefined above wherein said macromolecules are trimers.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof obtained by the methods asdefined above wherein said macromolecules are multimeric.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof obtainable by the methods asdefined above wherein said macromolecules are multimeric.

Another embodiment of the invention is a library, comprising one or morefunctional heavy chain antibodies, functional single domain antibodies,functional VH domains, or functional fragments thereof as defined above.

Another embodiment of the invention is a method to make a library,comprising at least one functional heavy chain antibody, functionalsingle domain heavy chain antibody, functional VH domain, or afunctional fragment thereof, comprising the steps:

-   -   introducing a restriction enzyme recognition site in the coding        region of the framework 4 region of a VH chain, whereby the        cutting site of said restriction enzyme recognition site is        situated in the CDR3 region,    -   cutting the nucleic acid molecule comprising said coding        sequence with said restriction enzyme,    -   ligating a double stranded primer to the VH encoding nucleic        acid molecule, restoring the CDR3 and so introducing an R amino        acid at position 103, said position determined by the Kabat        numbering, and    -   amplifying the ligated fragments.

Another embodiment of the invention is a method according to the above,whereby said restriction enzyme cut is situated within the last twocodons of the CDR3 coding region.

Another embodiment of the invention is a method according to the above,whereby said restriction enzyme creates a GA 3′ sticky end by cuttingbefore the first nucleotide of the codon coding for amino acid position101 and after the second nucleotide of codon coding for amino acidposition 101 on the complementary strand, said positions determinedaccording to the Kabat numbering.

Another embodiment of the invention is a method according to the above,whereby said restriction enzyme cut is situated within the last codon ofthe CDR3 coding region.

Another embodiment of the invention is a method according to the above,whereby said restriction enzyme is creating a CA-3′ sticky end bycutting before the second nucleotide of codon coding for amino acidposition 102 and after the third nucleotide of codon 102 on thecomplementary strand, said position determined according to the Kabatnumbering.

Another embodiment of the invention is a method according to the above,whereby said restriction enzyme is chosen from the group consisting ofBpmI, Eco57I, BsgI, Smu I, Fau I, Bse RI and Bfi I.

Another embodiment of the invention is a method to make a librarycomprising at least one functional heavy chain antibody, functionalsingle domain heavy chain antibody, functional VH domain, or functionalfragment thereof, comprising a step of amplification of nucleic acidstrands encoding a repertoire of immune or non-immune VHH antibodies,using a framework 1 specific primer as forward primer, and a back primerwhich anneals to said nucleic acid strands such that its 3′-terminalthree nucleotides are positioned over the codon of the nucleic acidstrands which encode amino acid position 103, the reverse-complement ofsaid 3′-terminal three nucleotides encoding R103, K103, Q103, F103,P103, G103 or S103, said position determined according to the Kabatnumbering.

Another embodiment of the invention is a method to make a librarycomprising at least one functional heavy chain antibody, functionalsingle domain heavy chain antibody, functional VH domain, or functionalfragment thereof, comprising a step of amplification of nucleic acidencoding a repertoire of immune or non-immune VHH antibodies orfragments thereof, using a framework 1 specific primer, as forwardprimer, and using one or more of SEQ ID NOs: 80 to 88 according to FIG.6 as back primers.

Another embodiment of the invention is a library, comprising at leastone functional heavy chain antibody, functional single domain heavychain antibody, functional VH domain, or functional fragment thereof,obtainable by the method according to any of claims as defined above.

Another embodiment of the invention is a library, comprising at leastone functional heavy chain antibody, functional single domain heavychain antibody, functional VH domain, or functional fragment thereof,obtained by the method as defined above.

Another embodiment of the invention is a library as defined abovewherein the methods use a single domain heavy chain library from humanor mouse.

Another embodiment of the invention is a library as defined abovewherein the methods use a single domain heavy chain library from camel.

Another embodiment of the invention is a library as defined abovewherein the methods use a single domain heavy chain library from sheep.

Another embodiment of the invention is a library as defined abovewherein the methods use a single domain heavy chain library from human.

Another embodiment of the invention is a library as defined abovewherein the methods use a single domain heavy chain library from mouse.

Another embodiment of the invention is a library as defined abovewherein the methods use a single domain heavy chain library from rat.

Another embodiment of the invention is a library as defined abovewherein the methods use a single domain heavy chain library from goat.

Another embodiment of the invention is a library as defined abovewherein the methods use a single domain heavy chain library from anyvertebrate species other than camel, human or mouse.

Another embodiment of the invention is a heavy chain antibody, afunctional single domain heavy chain antibody, a functional VH domain,or a functional fragment thereof, obtained by the method as definedabove for use in immunoassays.

Another embodiment of the invention is a recombinant DNA constructuseful for the expression of a polypeptide in a cell containing theconstruct, the construct comprising control sequences which regulatetranscription and translation of the said antibody in the cell and acoding sequence regulated by the control sequences, wherein the codingsequence comprises a DNA sequence of at least 21 bp in reading frame inthat the DNA sequence encodes a functional heavy chain antibody, afunctional single domain heavy chain antibody, a functional VH domain,or a functional fragment thereof as defined above or a polypeptide asdefined above.

Another embodiment of the invention is a recombinant DNA constructuseful for the expression of a polypeptide in a cell containing theconstruct, the construct comprising control sequences which regulatetranscription and translation of the said antibody in the cell and acoding sequence regulated by the control sequences, wherein the codingsequence comprises a DNA sequence of at least 42 bp in reading frame inthat the DNA sequence encodes a functional heavy chain antibody, afunctional single domain heavy chain antibody, a functional VH domain,or a functional fragment thereof as defined above or a polypeptide asdefined above.

Another embodiment of the invention is a recombinant DNA constructuseful for the expression of a polypeptide in a cell containing theconstruct, the construct comprising control sequences which regulatetranscription and translation of the said antibody in the cell and acoding sequence regulated by the control sequences, wherein the codingsequence comprises a DNA sequence of at least 63 bp in reading frame inthat the DNA sequence encodes a functional heavy chain antibody, afunctional single domain heavy chain antibody, a functional VH domain,or a functional fragment thereof as defined above or a polypeptide asdefined above.

Another embodiment of the invention is a recombinant DNA constructuseful for the expression of a polypeptide in a cell containing theconstruct, the construct comprising control sequences which regulatetranscription and translation of the said antibody in the cell and acoding sequence regulated by the control sequences, wherein the codingsequence comprises a DNA sequence of at least 83 bp in reading frame inthat the DNA sequence encodes a functional heavy chain antibody, afunctional single domain heavy chain antibody, a functional VH domain,or a functional fragment thereof as defined above or a polypeptide asdefined above.

Another embodiment of the invention is a recombinant DNA constructuseful for the expression of a polypeptide in a cell containing theconstruct, the construct comprising control sequences which regulatetranscription and translation of the said antibody in the cell and acoding sequence regulated by the control sequences, wherein the codingsequence comprises a DNA sequence of at least 150 bp in reading frame inthat the DNA sequence encodes a functional heavy chain antibody, afunctional single domain heavy chain antibody, a functional VH domain,or a functional fragment thereof as defined above or a polypeptide asdefined above.

Another embodiment of the invention is a recombinant DNA constructuseful for the expression of a polypeptide in a cell containing theconstruct, the construct comprising control sequences which regulatetranscription and translation of the said antibody in the cell and acoding sequence regulated by the control sequences, wherein the codingsequence comprises a DNA sequence of at least 240 bp in reading frame inthat the DNA sequence encodes a functional heavy chain antibody, afunctional single domain heavy chain antibody, a functional VH domain,or a functional fragment thereof as defined above or a polypeptide asdefined above.

Another embodiment of the invention is a recombinant DNA constructuseful for the expression of a polypeptide in a cell containing theconstruct, the construct comprising control sequences which regulatetranscription and translation of the said antibody in the cell and acoding sequence regulated by the control sequences, wherein the codingsequence comprises a DNA sequence of at least 300 bp in reading frame inthat the DNA sequence encodes a functional heavy chain antibody, afunctional single domain heavy chain antibody, a functional VH domain,or a functional fragment thereof as defined above or a polypeptide asdefined above.

Another embodiment of the invention is a nucleic acid comprising a DNAsequence encoding a functional heavy chain antibody, a functional singledomain heavy chain antibody, a functional VH domain, or a functionalfragment thereof as defined above or a polypeptide as defined above.

Another embodiment of the invention is a nucleic acid having anucleotide sequence which is at least 65% identical to the sequence asdefined above.

Another embodiment of the invention is a vector comprising a nucleicacid sequence as defined above.

Another embodiment of the invention is a host cell comprising anintegrated or episomal copy of a nucleic acid molecule as defined above,or a vector as defined above.

Another embodiment of the invention is the host cell as used above,wherein said host cell is a yeast, bacterial, insect, fungal, plant ormammalian cell.

Another embodiment of the invention is a method for producing afunctional heavy chain antibody, a functional single domain heavy chainantibody, a functional VH domain, or a functional fragment thereof asdefined above or a polypeptide as defined above, comprising:

(a) culturing host cells comprising a nucleic acid as defined above,under conditions allowing the expression of the polypeptide, and,

(b) recovering the produced polypeptide from the culture.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof as defined above or apolypeptide as defined above, or nucleic acid as defined above for thepreparation of a medicament.

Another embodiment of the invention is a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof as defined above or apolypeptide as defined above, or nucleic acid as defined above for thepreparation of a medicament for the treatment of a disease related toasthma, rhinoconjunctivitis, allergic disorders, acute allograftrejection, Crohn's disease and ulcerative colitis.

Another embodiment of the invention is a pharmaceutical compositioncomprising a functional heavy chain antibody, a functional single domainheavy chain antibody, a functional VH domain, or a functional fragmentthereof as defined above or a polypeptide as defined above, or nucleicacid as defined above, optionally in combination with a suitableexcipient.

Another embodiment of the invention is the use of a functional heavychain antibody, a functional single domain heavy chain antibody, afunctional VH domain, or a functional fragment thereof as defined aboveor a polypeptide as defined above, or nucleic acid as defined above inthe diagnosis of a disease related to asthma, rhinoconjunctivitis,allergic disorders, acute allograft rejection, Crohn's disease andulcerative colitis.

Another embodiment of the invention is the use of a functional heavychain antibody, a functional single domain heavy chain antibody, afunctional VH domain, or a functional fragment thereof as defined aboveor a polypeptide as defined above for the purification of a protein.

Another embodiment of the invention is a kit for the diagnosis of apathological condition or a susceptibility to a pathological conditionin a subject comprising a nucleic acid as defined above, a functionalheavy chain antibody, a functional single domain heavy chain antibody, afunctional VH domain, or a functional fragment thereof as defined aboveor a polypeptide as defined above.

Another embodiment of the invention is a method for diagnosing apathological condition or a susceptibility to a pathological conditionin a subject comprising the steps of:

(a) determining the presence or absence of a mutation in the nucleicacid as defined above, including mutations in the genomic and regulatorysequences of said nucleic acid, in a biological sample, and

(b) diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or absence of saidmutation.

Another embodiment of the invention is a method for diagnosing apathological condition or a susceptibility to a pathological conditionin a subject comprising the steps of:

(a) determining the presence or amount of the nucleic acid as definedabove or expression of a functional heavy chain antibody, a functionalsingle domain heavy chain antibody, a functional VH domain, or afunctional fragment as defined above or a polypeptide as defined abovein a biological sample, and,(b) diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or amount of said nucleicacid or expression of said functional heavy chain antibody, functionalsingle domain heavy chain antibody, functional VH domain, functionalfragment thereof or polypeptide.

Another embodiment of the invention is a drug screening assay forscreening test compounds which interact with a functional heavy chainantibody, a functional single domain heavy chain antibody, a functionalVH domain, or a functional fragment thereof as defined above or apolypeptide as defined above, comprising:

(a) combining a functional heavy chain antibody, a functional singledomain heavy chain antibody, a functional VH domain, or a functionalfragment thereof as defined above or a polypeptide as defined above witha test compound, under conditions which allow for interaction of thetest compound to said functional heavy chain antibody, functional singledomain heavy chain antibody, functional VH domain, functional fragmentthereof or polypeptide, to form a complex, and,(b) detecting the formation of a complex, in which the ability of thetest compound to interact with the said functional heavy chain antibody,functional single domain heavy chain antibody, functional VH domain, orfunctional fragment thereof, is indicated by the presence of the testcompound in the complex.

Another embodiment of the invention is the product or compoundidentifiable by the assay as defined above.

Another embodiment of the invention is nucleic acid comprising thesequence SEQ ID NO: 80 according to FIG. 6.

Another embodiment of the invention is nucleic acid comprising thesequence SEQ ID NO: 81 according to FIG. 6.

Another embodiment of the invention is nucleic acid comprising thesequence SEQ ID NO: 82 according to FIG. 6.

Another embodiment of the invention is nucleic acid comprising thesequence SEQ ID NO: 83 according to FIG. 6.

Another embodiment of the invention is nucleic acid comprising thesequence SEQ ID NO: 84 according to FIG. 6.

Another embodiment of the invention is nucleic acid comprising thesequence SEQ ID NO: 85 according to FIG. 6.

Another embodiment of the invention is nucleic acid comprising thesequence SEQ ID NO: 86 according to FIG. 6.

Another embodiment of the invention is nucleic acid comprising thesequence SEQ ID NO: 87 according to FIG. 6.

Another embodiment of the invention is nucleic acid comprising thesequence SEQ ID NO: 88 according to FIG. 6.

Another embodiment of the invention is a nucleic acid having anucleotide sequence which is at least 65% identical to the sequence asdefined above.

Another embodiment of the invention is the use of a nucleic acid asdefined above in a method to produce one or more functional heavy chainantibodies, functional single domain heavy chain antibodies, functionalVH domains, or functional fragments thereof.

The antibodies of the above embodiments are functional and as suchexhibit improved properties, for example, expression levels, stability,affinity and solubility over antibodies in which the characterisingfeatures are absent. It is known in the art that the production ofantibodies, for example by bacterial overexpression techniques, in phagedisplay libraries, for screening libraries, is difficult due to theproperties of the antibody or fragments thereof.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, the inventors have found that a heavy chain, carrying amutation at position 103, possibly combined with a mutation at position108 behaves as a functional heavy chain antibody (HCAb), even if it doesnot carry the VHH hallmark amino acids at positions 37, 44, 45 and 47.In particular, the inventors have found that a mutation wherein theamino acid residue corresponding to position 103 (Kabat numbering) ismutated to an amino acid selected among arginine, glycine, proline,serine, leucine, glutamine or lysine can compensate for the loss of thecritical hallmark amino acid at position 45, whereby the charged aminoacid or the cysteine at position 45, may be changed in any other aminoacid, but preferably into a leucine. Prior to an aspect of the presentinvention, heavy chains having said amino acids at positions at 45 and103 were considered by the person skilled in the art as part of aclassical 4-chain antibody complex (Harmsen et al. 2000) and not as afunctional heavy chain antibody. Moreover, for some antibodies havingresidues which characterise one aspect of the present invention, thepresence of a light chain has been described (Anker et al., 1990;Chukwuocha at al., 1999), clearly demonstrating that these antibodieswere not functional heavy chain antibodies.

Surprisingly, the inventors were able to show that an amino acid atposition 103 selected among arginine, glycine, proline, serine or lysineincreases the solubility of the heavy chain, while it may disrupt thepossibility to interact with a light chain. Therefore, such a heavychain molecule behaves like a functional heavy chain antibody, evenwithout the hallmark amino acid for the functional HCAb at position 45.

The invention relates to a functional HCAb, comprising an amino acid,which is neither a charged amino acid nor a cysteine at position 45, andcomprising an amino acid chosen from the group consisting of arginine(R), glycine (G), lysine (K), serine (S) and proline (P) at position103, possibly combined with glutamine (Q) at position 108 according tothe Kabat numbering. In one embodiment of the invention, the amino acidat position 45 is a L. In another embodiment, the amino acid at position103 is an R. In another embodiment, the amino acid at position 103 is anR and the amino acid at position 108 is a Q.

In one embodiment of the invention embodiment, the functional HCAb orfragment thereof according to the invention is an artificial mutant. Anartificial mutant, as used here, means that the change is introducedintentionally and differs from the sequence found in the naturalsituation. Said artificial mutant may be derived from a variable domain(designated VH) of a heavy polypeptide chain of an immunoglobulinwherein the amino acid residue corresponding to position 103 (Kabatnumbering) is mutated to an amino acid selected from the groupconsisting of R, G, K, S and P, possibly in combination with a mutationwherein the amino acid residue corresponding to position 108 (Kabatnumbering) is mutated to Q. In another embodiment, said artificialmutant is derived from a variable domain (designated VH) of a heavypolypeptide chain of an immunoglobulin wherein the amino acid residuecorresponding to position 103 (Kabat numbering) is mutated to arginine.In another embodiment, said artificial mutant is derived from a variabledomain (designated VH) of a heavy polypeptide chain of an immunoglobulinwherein the amino acid residue corresponding to position 103 (Kabatnumbering) is mutated to arginine and the amino acid residuecorresponding to position 108 (Kabat numbering) is mutated to glutamine.Alternatively, said artificial mutant may be derived from a variabledomain (designated VHH) of a heavy polypeptide chain of a heavy chainantibody wherein the hallmark amino acid residue at position 45 ismutated to a Leucine, possibly in combination with a mutation of one ormore of the other hallmark amino acids at position 37, 44 and 47 toVal37, Gly44 and Trp47. One embodiment is an artificial mutant whereinall hallmark amino acids at position 37, 44, 45 and 47 are mutated toVal37, Gly44, Leu45 and Trp47.

An artificial mutant according to the invention comprises a polypeptidesequence derived from the VH domain and encompasses within thispolypeptide sequence RGQGTQ (SEQ ID NO: 13) or alternatively sequenceRGKGTQ (SEQ ID NO: 14).

Another artificial mutant according to the invention comprises apolypeptide sequence derived from the VHH domain and encompasses withinthis polypeptide sequence VXXXXXXGLXW (SEQ ID NO: 15), whereby X can beany amino acid.

Another artificial mutant according to the invention comprises apolypeptide sequence derived from the VH domain and encompasses withinthis polypeptide sequence RGQGTQ (SEQ ID NO: 13), wherein R of saidsequence is at position 103 according to the Kabat numbering. Anotherartificial mutant according to the invention comprises a polypeptidesequence derived from the VH domain and encompasses within thispolypeptide sequence RGKGTQ (SEQ ID NO: 14), wherein R of said sequenceis at position 103 according to the Kabat numbering.

Another artificial mutant according to the invention comprises apolypeptide sequence derived from the VHH domain and encompasses withinthis polypeptide sequence VXXXXXXGLXW (SEQ ID NO: 15), wherein V of saidsequence is at position 37 according to the Kabat numbering, whereby Xcan be any amino acid.

Another artificial mutant according to the invention comprises apolypeptide sequence derived from the VH domain and encompasses withinthis polypeptide sequence LGQGTQVTVSS (SEQ ID NO: 16), wherein L of saidsequence is at position 103 according to the Kabat numbering.

Another artificial mutant according to the invention comprises apolypeptide sequence derived from the VH domain and encompasses withinthis polypeptide sequence QGQGTGVTVSS (SEQ ID NO: 17), wherein L of saidsequence is at position 103 according to the Kabat numbering.

Another artificial mutant according to the invention comprises apolypeptide sequence derived from the VH domain and encompasses withinthis polypeptide sequence PGQGTQVTVSS (SEQ ID NO: 18), wherein L of saidsequence is at position 103 according to the Kabat numbering.

Another artificial mutant according to the invention comprises apolypeptide sequence derived from the VH domain and encompasses withinthis polypeptide sequence SSQGTQVTVSS (SEQ ID NO: 19), wherein L of saidsequence is at position 103 according to the Kabat numbering.

Said SEQ ID NOs: 13 to 19 are those cited in FIG. 6.

The artificial mutant according to the invention is derived from a VHdomain, or a VHH domain, meaning that in accordance with the presentinvention it can be isolated from said domain by introducing saidmutations, or it can be synthesized, including chemically synthesized orexpressed especially by recombinant techniques, including in host cells,starting from the knowledge of the polypeptide sequence of said VHdomains or VHH domains and the position of the mutations to beintroduced. More generally, it can be prepared by any method madeavailable for the preparation of polypeptide chains.

The polypeptide chain derived from the VH gene and having the featuresas defined here above may be obtained by methods involving site-directedmutagenesis or PCR (using primers carrying said mutation), starting froma conventional VH-D-J gene, especially one obtained from a library. Arelevant method is that described by Hemsley et al. (1989). The presentinvention thus provides the possibility to generate soluble mutantsingle domain antibody fragments that originate from a VH-D-J gene. Themethod of Hemsley, however, requires that the sequence of the gene to bemutated is known, at least in the region where the mutation has to beintroduced. As a position to be mutated, residue 103, is adjacent to avariable region, the method of Hemsley et al. (1989) is not suitable forthe introduction of a mutation in VHs with unknown variable regions, andan adapted method has to be applied in this case.

Alternatively, the polypeptide chain derived from the VHH and having thefeatures as defined here above may be obtained by methods involvingsite-directed mutagenesis or PCR (using primers carrying said themutation), starting from a HCAb VHH, especially one obtained from alibrary. As these mutations are situated in a conserved frameworkregion, the method of Hemsley et al. (1989) can be applied. In thatcase, the present invention provides the possibility to humanize singledomain antibody fragments that originate from a VHH-D-J gene.Humanization may comprise the replacement of one or more of the VHHhallmark amino acids at position 37, 44, 45 and 47 into the conservedhuman residues Val37, Gly44, Leu 45 and Trp47. However, to introduce thecompensating mutation at position 103, the sequence of the adjacentvariable region should be known, as discussed above.

The invention relates further to a method to solubilize single domainheavy chain fragment derived from conventional 4-chain immunoglobulins.Indeed, the inventors have shown that the presence at position 103 of ahydrophilic amino acid residue, especially of a residue selected amongarginine, glycine, proline, serine or lysine renders the resultingpolypeptide derived from the VH more soluble with respect to the samepolypeptide having a tryptophan residue at position 103. This effect mayeven be enhanced by replacing the amino acid at position 108 by aglutamine.

Another aspect of the invention is a method to “humanize” a camelidheavy chain antibody, said method comprising at least the replacement ofthe camelid hallmark amino acid at position 45, possibly combined with areplacement of one or more of the other hallmark amino acids at position37, 44 and 47. Humanizing, as used here, means that one or more of thecamelid hallmark amino acids in the HCAb are replaced by their humancounterpart as found in the human consensus sequence, without that saidheavy chain antibody is losing its typical character, i.e. thehumanization does not significantly affect the antigen binding capacityof the resulting HCAb or fragment thereof.

Another aspect of the invention is a functional single domain heavychain antibody fragment, obtainable by the method according to theinvention. Still another aspect of the invention is a functional singledomain heavy chain antibody fragment, obtained by the method accordingto the invention. Still another aspect of the invention is a functionalhumanized single domain heavy chain antibody fragment, obtainable by themethod according to the invention. Still another aspect of the inventionis a functional humanized single domain heavy chain antibody fragment,obtained by the method according to the invention.

Another aspect of the invention is a library, comprising one or morefunctional single domain heavy chain antibody fragments according to theinvention. In one embodiment of the present invention, said librarycomprises at least 100, or at least 1000, or at least 10 000, or atleast 100 000, or at least 1 000 000 functional single domain heavychain antibody fragments. Such a library has the advantage that it iscomposed of soluble molecules, contrary to a possible library ofclassical VH molecules, which would be insoluble and non-functional(Nutall et al., 2000). Whereas for one single VH, the problem may besolved by denaturation, followed by refolding and resolubilization, thisprocedure is not possible in case of the creation of a complex library,especially not when said creation is followed by the selection ofdomains with varying antigen binding specificities. Indeed, generationof insoluble and therefore “sticky” scaffold protein during selectionprocedures can lead to false positives through non-specific binding toantigen by hydrophobic patches on the displayed domain. Several attemptshave been made to overcome this aggregation problem (Pessi et al, 1993;Quiocho, 1993; Dimasi et al., 1997), but till now, the attempts ofproducing single domain VH libraries with acceptable solubilitycharacteristics have not been successful. Therefore, the soluble singledomain heavy chain antibody fragment according to the invention allowfor the first time the efficient construction of a library comprisingfunctional single chain antibody fragments.

As the functional single domain heavy chain antibodies according to theinvention do normally not occur in nature, such a library cannot be madeby direct amplification of the messenger RNA, but has to be made by theuse of mutagenic primers. Although in principle, a VHH backbone may beused as starting material, this has several drawbacks, as not only thecamelid hallmark amino acids have to be mutated, but the compensatingmutation at position 103 has to be introduced. For this reason, it ispreferable to construct the library starting from a human VH mRNAlibrary. From such a library, potent antigen binders may be retrieved.Contrary to the VHH situation, starting from a VH backbone, only themutation at position 103 has to be introduced.

The problem to make such library is that the 3′-end primer used toamplify the VH by RT-PCR needs to be mutagenic for the codon 103, andthe primer should extend for at least 4-5 nucleotides towards its 3′-endto anneal perfectly to the template. However, since this region is partof the CDR3, such a primer will contain too much degeneracies so that noDNA amplification will be achieved. As a consequence, making afunctional single domain heavy chain library, according to the inventionis far from obvious.

Therefore another aspect of the invention is a method to make a library,comprising at least one functional single chain library, according tothe invention, comprising

-   -   introducing a restriction enzyme recognition site in the coding        region of the framework 4 region of a VH or VHH chain, whereby        the cutting site of said restriction enzyme recognition site is        situated in the CDR3 region    -   cutting the nucleic acid molecule comprising said coding        sequence with said restriction enzyme    -   ligating a double stranded primer to the remaining V encoding        nucleic acid molecules, restoring the CDR3 and framework 4        codons and introducing the 103 mutation in the framework 4    -   amplifying the ligated fragments.

One embodiment comprises said method whereby the method is carried outon a pool of coding sequences, such as a pool of mRNA as well as on oneisolated coding sequence. Another embodiment comprises said methodwhereby the method is carried out on one isolated coding sequence, and asynthetic library is generated by randomizing one or more codons of oneor more of the CDR loops. Alternatively, a library may be generated bygrafting camelid CDR loops on the mutated framework, comprising the 103Rmutation.

In another embodiment, the restriction enzyme cut is situated within thelast codon or within the last two codons of the CDR3 coding region. Oneembodiment comprises said method, whereby said enzyme is creating ablunt end at the CDR3—framework 4 junction. Another embodiment comprisessaid method, whereby said enzyme creates a CA 3′ sticky end by cuttingbefore the second nucleotide of codon 102 and after the secondnucleotide of codon 102, according to the Kabat numbering. Indeed, inmost VH and VHH's, there is a conserved tyrosine (Y) at position 102.This amino acid is most frequently encoded by TAC. Another embodimentcomprises said method, whereby said enzyme creates a GA 3′ sticky end bycutting before the first nucleotide of codon 101 and after the secondnucleotide of codon 101, according to the Kabat numbering. Indeed, inmost human VH, there is a conserved aspartic acid (D) at position 101, acharged amino acid that is important for the CDR3 loop structure. Thisamino acid is encoded by either GAC or GAT. By creating a GA 3′ stickyend, the conserved codon may be restored by the ligation to the doublestranded primer. In that case, the codon 102 may be either randomized orfixed, by ligation of the primer. One embodiment comprises said methodabove, whereby said restriction site is Bpm I. Another embodimentcomprises said method, whereby said restriction site is Eco57 I. Anotherembodiment comprises said method, whereby said restriction site is BsgI. An embodiment comprises said method, whereby said restriction site isFau I. Another embodiment comprises said method, whereby saidrestriction site is Smu I. Another embodiment comprises said method,whereby said restriction site is Bse R1.

Another embodiment comprises said method, whereby the restriction siteis BfiI, introduced in such a way that the enzyme cuts at the CDR3junction in the upper strand, and between the first and the secondnucleotide of CDR3, adjacent to the framework 4 in the lower strand. Inthe latter case, the CDR3 and framework 4 regions may be restored byligation with a double stranded primer consisting of the framework 4coding region for the upper strand, and the complementary strandthereof, with either a TG 3′ overhang, or a TG-3′ overhang and an extracodon such as GTG, or TAC before the Trp103 codon.

Still another aspect of the inventions is a library obtainable by theinvention, comprising one or more functional single domain heavy chainantibody fragments. Still another aspect of the invention is a library,obtained by the invention, comprising one or more functional singledomain heavy chain antibody fragments.

In one embodiment, said library comprises at least 100, in anotherembodiment at least 1000, in another embodiment at least 10 000, inanother embodiment at least 100 000, in another embodiment at least 1000 000 functional single domain heavy chain antibody fragments.

Surprisingly, we have found that a significant fraction of the camelidantibodies comprises functional heavy chain antibodies according to theinvention, contrary to what is assumed by the person skilled in the art.This significant fraction represent a new class of functional heavychain antibodies. It would not be obvious, therefore, to a skilledartisan that a functional heavy chain antibody and/or a functionalsoluble single domain heavy chain antibody fragment can be isolateddirectly from a mRNA preparation from camelids, and this material can beused as starting material for the preparation of a functional solublesingle domain heavy chain antibody fragment library according to theinvention. As a consequence, another aspect of the invention is alibrary obtained by specific amplification and cloning of the new classof functional heavy chain antibodies described in this invention, andwhich have more homology to human antibodies than the class of VHH withthe hydrophilic residues in FR2. In order to obtain this new class ofVHH from a repertoire of immune or non-immune antibodies, specificprimers for amplification were designed, that anneal preferentially togenes encoding VHH with Arginine, Lysine, Glutamine, Phenylalanine,Proline, Glycine, Tryptophan or Serine as residue 103. To accomplishspecific annealing the 3′ site of the primer ends exactly at the firstnucleotide of the codon coding for residue 103, which in the new classof VHH is different from the Tryptophan 103 containing VHH fragments.

The following primers were designed:

primer 1 (R103): (SEQ ID NO: 80) 5′-GAG TCA TTC TCG ACT TGC GGC CGC TGAGGA GAC GGT GAC CTG GGT CCC CTG GCC (A/T/C/G)CG-3′ primer 2 (R103): (SEQID NO: 81) 5′-GAG TCA TTC TCG ACT TGC GGC CGC TGA GGA GAC GGT GAC CTGGGT CCC CTG GCC (C/T)CT-3′ primer 3 (K103): (SEQ ID NO: 82) 5′-GAG TCATTC TCG ACT TGC GGC CGC GCT GGA GAC GGT GAC CTG GGT CCC CTG GCC(T/C)TT-3′ primer 4 (Q103): (SEQ ID NO: 83) 5′-GAG TCA TTC TCG ACT TGCGGC CGC TGA GGA GAC GGT GAC CTG GGT CCC CTG GC(C/G) (C/T)TG-3′ primer 5(L103): (SEQ ID NO: 84) 5′-GAG TCA TTC TCG ACT TGC GGC CGC TGA GGA GACGGT GAC CTG GGT CCC CTG GCC (A/G/C/T)AG-3′ primer 6 (F103): (SEQ ID NO:85) 5′-GAG TCA TTC TCG ACT TGC GGC CGC TGA GGA GAC GGT GAC CTG GGT CCCCTG GCC (A/G)AA-3′ primer 7 (G103): (SEQ ID NO: 86) 5′-GAG TCA TTC TCGACT TGC GGC CGC TGA GGA GAC GGT GAC CTG GGT CCC CCC CGG (A/G/C/T)CC-3′primer 8 (S103): (SEQ ID NO: 87) 5′-GAG TCA TTC TCG ACT TGC GGC CGC TGAGGA GAC GGT GAC CTG GGT CCC CTG (A/G/C/T)GA (A/G/C/T)GA-3′ primer 9(P103): (SEQ ID NO: 88) 5′-GAG TCA TTC TCG ACT TGC GGC CGC TGA GGA GACGGT GAC CTG GGT CCC CTG CTG (A/G/C/T)GG-3′ primer 10 (Y103): (SEQ ID NO:89) 5′-GAG TCA TTC TCG ACT TGC GGC CGC TGA GGA GAC GGT GAC CTG GGT CCCCTG GCC (A/G)TA-3′

Specific amplification was carried out using a Framework 1 specificprimer, that is 5′ linked to a SfiI site, as forward primer, and a poolof primers 1-10 as back primers for the amplification of functionalsoluble single domain heavy chain antibody fragments according to theinvention. The resulting material was cut with SfiI and NotI or BstEIIand the resulting fragment is cloned into pHen4.

Still another aspect of the invention is the use of a functional heavychain antibody according to the invention or a functional soluble singledomain heavy chain antibody fragment according to the invention for thepreparation of a medicament. Still another aspect of the invention is apharmaceutical composition, comprising a functional heavy chainantibody, according to the invention, or comprising a functional solublesingle domain heavy chain antibody fragment, according to the invention,optionally in combination with a suitable excipient. Indeed, antibodiesmay be used in the treatment of several diseases, such as, as anon-limiting example, asthma and rhinoconjunctivitis (Botger et al,2002), allergic disorders (Babu et al, 2001), acute allograft rejection(Sollinger et al, 2001), Crohn's disease (Hommes et al, 2002) andulcerative colitis (Gordon et al, 2002). The functional soluble singledomain heavy chain antibody fragment may have a significant advantagedue to their small size and their solubility.

Another aspect of the invention is the use of a functional heavy chainantibody according to the invention or a functional soluble singledomain heavy chain antibody fragment according to the invention indiagnosis. Diagnostic methods, using antibodies are known to the personskilled in the art and include, as a non-limiting example ELISA and RIAmethods. The antibodies according to the invention do have severaladditional advantages in these assays, due to their stability and thefact that they can be fixed on a solid support without significant lossof activity. The latter characteristic makes them specially suitable forcoating of surfaces, as may be desirable in several immunologicaldetection techniques, including their use in microarrays.

Still another aspect of the invention is the use of a functional heavychain antibody according to the invention or a functional soluble singledomain heavy chain antibody fragment according to the invention in thepurification of proteins and other molecules. Purification methods suchas, as a non-limiting example, immunochromatography are known to theperson skilled in the art. The antibodies according to the invention dohave several additional advantages in such purification methods, due totheir stability, that may guarantee a long lifetime of the purificationcarrier, and due to the fact that they can be fixed on a solid supportwithout significant loss of activity.

DEFINITIONS

The following definitions are set forth to illustrate and define themeaning and scope of various terms used to describe the inventionherein.

“Derivatized” as used herein in reference to a polypeptidicmacromolecule means comprising derivatized amino acids. For example,homo-phenylalanine, citrulline, and noreleucine are consideredderivatized amino acids for the purposes of the invention. Derivatizedamino acids also include imino acid residues such as proline andhydroxyproline. In addition, any amino acid representing a component ofthe variant proteins of the present invention, replaced by the sameamino acid but of the opposite chirality, is considered derivatized.Thus, any amino acid naturally occurring in the L-configuration (whichmay also be referred to as the R or S, depending upon the structure ofthe chemical entity) may be replaced with an amino acid of the samechemical structural type, but of the opposite chirality, generallyreferred to as the D-amino acid but which can additionally be referredto as the R or the S, depending upon its composition and chemicalconfiguration. Such derivatives have the property of greatly increasedstability, and therefore are advantageous in the formulation ofcompounds which may have longer in vivo half lives, when administered byoral, intravenous, intramuscular, intraperitoneal, topical, rectal,intraocular, or other routes.

In the preferred embodiment, the derivatized amino acids are in the (S)or L-configuration or the (S) or D-configuration. Derivatized aminoacids may be used, for example, to prevent or retard in vivodegradations. Proteins including non-naturally occurring amino acids maybe synthesized or in some cases, made recombinantly; see van Hest etal., FEBS Lett 428:(1-2) 68-70 May 221998 and Tang et al., Abstr. PapAm. Chem. S218:U138-U138 Part 2 Aug. 22, 1999, both of which areexpressly incorporated by reference herein.

Aromatic amino acids may be replaced with D- or L-naphylalanine, DM orL-Phenylglycine, D- or L-2-thieneylalanine, D- or L-1-, 2-, 3- or4-pyreneylalanine, D- or L-3-thieneylalanine, D- orL-(2-pyridinyl)-alanine, D- or L-(3-pyridinyl)-alanine, D- orL-(2-pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine,D-(trifluoromethyl)-phenylglycine, D-(trifluoromethyl)-phenylalanine,D-p-fluorophenylalanine, D- or L-p-biphenylphenylalanine, D- orL-p-methoxybiphenylphenylalanine, D- or L-2-indole(alkyl)alanines, andD- or L-alkylainines where alkyl may be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, non-acidic amino acids, of C1-C20.

Acidic amino acids can be regarded as derivatized when they aresubstituted with non-carboxylate amino acids while maintaining anegative charge, and derivatives or analogs thereof, such as thenon-limiting examples of (phosphono)alanine, glycine, leucine,isoleucine, threonine, or serine; or sulfated (e.g., —SO3H) threonine,serine, or tyrosine.

Other substitutions may include unnatural hyroxylated amino acids. Otherderivatives may made by combining “alkyl” with any natural amino acid.The term “alkyl” as used herein refers to a branched or unbranchedsaturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl,tetradecyl, hexadecyl, eicosyl, tetracisyl and the like. Alkyl includesheteroalkyl, with atoms of nitrogen, oxygen and sulfur. Preferred alkylgroups herein contain 1 to 12 carbon atoms. Basic amino acids may besubstituted with alkyl groups at any position of the naturally occurringamino acids lysine, arginine, ornithine, citrulline, or(guanidino)-acetic acid, or other (guanidino)alkyl-acetic acids, where“alkyl” is defined as above. Nitrile derivatives (e.g., containing theCN-moiety in place of COOH) may also be substituted for asparagine orglutamine, and methionine sulfoxide may be substituted for methionine.Methods of preparation of such peptide derivatives are well known to oneskilled in the art.

In addition, any amide linkage in any of the variant polypeptides can bereplaced by a ketomethylene moiety. Such derivatives are expected tohave the property of increased stability to degradation by enzymes, andtherefore possess advantages for the formulation of compounds which mayhave increased in vivo half lives, as administered by oral, intravenous,intramuscular, intraperitoneal, topical, rectal, intraocular, or otherroutes.

Additional amino acid modifications of amino acids of variantpolypeptides of to the present invention may include the following:Cysteinyl residues may be reacted with α-haloacetates (and correspondingamine), such as 2-chloroacetic acid or chloroacetamide, to givecarboxymethyl or carboxyamidomethyl derivatives.

Cysteinyl residues may also be derivatized by reaction with compoundssuch as bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, P-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues may be derivatized by reaction with compounds such asdiethylprocarbonate e.g., at pH 5.5 to 7.0 because this agent isrelatively specific for the histidyl side chain, and para-bromophenacylbromide may also be used, e.g., where the reaction is preferablyperformed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues may be reacted with compounds suchas succinic or other carboxylic acid anhydrides. Derivatization withthese agents is expected to have the effect of reversing the charge ofthe lysinyl residues.

Other suitable reagents for derivatizing α-amino-containing residuesinclude compounds such as imidoesters e.g., as methyl picolinimidate;pyridoxal phosphate; pyridoxal; chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; andtransaminase-catalyzed reaction with glyoxylate. Arginyl residues may bemodified by reaction with one or several conventional reagents, amongthem phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrinaccording to known method steps. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group. The specific modification of tyrosyl residues perse is well-known, such as for introducing spectral labels into tyrosylresidues by reaction with aromatic diazonium compounds ortetranitromethane.

N-acetyll midizol and tetranitromethane may be used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Carboxyl sidegroups (aspartyl or glutamyl) may be selectively modified by reactionwith carbodiimides (R′—N—C—N—R′) such as1-cyclohexyl-3-(2-morpholiny-1-(4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermoreaspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions. Glutaminyl andasparaginyl residues may be frequently deamidated to the correspondingglutamyl and aspartyl residues. Alternatively, these residues may bedeamidated under mildly acidic conditions. Either form of these residuesfalls within the scope of the present invention.

“Functional” in reference to a heavy chain antibody, a single domainheavy chain antibody, a VH domain or fragments thereof means that thesame retains a significant binding (dissociation constant in themicromolar range) to its epitope, compared with its binding in vivo, andthat it shows no or limited aggregation (soluble and non-aggregatedabove 1 mg/ml), so allowing the use of the antibody as a binder.

“Functionalized” in reference to a heavy chain antibody, a single domainheavy chain antibody, or fragments thereof means to render said heavychain antibody, a single domain heavy chain antibody, or fragmentsthereof functional.

By “fragments thereof” as used herein, is meant a portion correspondingto more than 95% of the sequence, more than 90% of the sequence of, morethan 85% of the sequence of, more than 80% of the sequence of, more than75% of the sequence of, more than 70% of the sequence of, more than 65%of the sequence of, more than 60% of the sequence of, more than 55% ofthe sequence of, or more than 50% of the sequence of.

“Coding sequence” is a nucleotide sequence, which is transcribed intomRNA and/or translated into a polypeptide when placed under the controlof appropriate regulatory sequences. The boundaries of the completecoding sequence are determined by a translation start codon at the5′-terminus and a translation stop codon at the 3′-terminus. However,coding sequence as used here is not limited to the complete codingsequence, but includes fragments thereof; such fragments are alsoindicated as coding region. A coding sequence can include, but is notlimited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA,while introns may be present as well under certain circumstances.

“Human hallmark amino acids” as used herein in reference to thehumanization of non-human antibodies are Val37, Gly44, Leu45, Trp47,positions determined according to the Kabat numbering.

“Nucleotide sequence” “DNA sequence” “nucleic acid molecule(s)” or“nucleic acid” as used herein refers to a polymeric form of nucleotidesof any length, either ribonucleotides or deoxyribonucleotides. This termrefers only to the primary structure of the molecule. Thus, this termincludes double- and single-stranded DNA, and RNA. It also includesknown types of modifications, for example, methylation, “caps”substitution of one or more of the naturally occurring nucleotides withan analog.

“Upper strand” of a DNA sequence is the strand that comprise the DNAversion of the codons as they occur in the mRNA, lower strand is thestrand with the anticodons, that is used as template to synthesize themRNA.

“VH domain” as used herein means the variable domain of H-chain of aconventional four-chain antibody.

“VHH domain” as used herein means variable domain of the H-chain of aconventional, (i.e. immunologically functional) HCAb.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1-1 and 1-2: Nucleotide and translated amino acid sequences ofwild type anti-b-lactamase VHH TEM04 (A; SEQ ID NO:11 and SEQ ID NO:12)and anti-carcinoembryonic antigen VHH CEA71 (B; SEQ ID NO:92 and SEQ IDNO:93).

FIG. 2: Western blot analysis of R103W mutant and wild type VHH from (A)anti-β-lactamase VHH TEM04 and (B) anti-carcinoembryonic antigen VHHCEA71. From each clone three cultures were induced and used foranalysis. As positive control the anti-lysozyme VHH cAblys3 was used.

FIG. 3: Coomassie stained 15% SDS PAGE of IMAC purified R103W mutant andwild type VHH. The slower migrating product in the two lanes on the leftis β-lactamase, which was complexed to the VHH.

FIG. 4: Antigen binding determined by ELISA of VHH CEA71 wild type (WT)and R103W mutant.

FIG. 5: Examination of the solubility characteristics of VHH CEA72 (wildtype and R103W mutant) by determining the concentration of ammoniumsulfate at which the fragment started to precipitate. The amount of VHHin the supernatant was measured (A) by protein content with OD280, or(B) by antigen binding in ELISA.

FIG. 6-1, 6-2, 6-3: SEQ ID NOS: 1 to 10 and 20 to 79: sequences of heavychain antibody, single domain antibody, a VH domain, or a fragmentthereof, wherein amino acids at positions indicated are substituted byamino acids indicated, said positions determined according to the Kabatnumbering.

EXAMPLES Example 1 Camelization of Isolated VH1

Classically, a recombinant VH domain is isolated from scFv libraries.Such VH domains usually originate from a cloning artifact for example bythe cloning of VH instead of VH-VL, or they may originate from a generecombination within the clone, due for example to instability of thelinker sequences, resulting in the deletion of the VL gene fragment.These molecules are normally difficult to work with because of their lowexpression yields in, for example, bacterial and other expressionsystems and their low solubility. The inventors show that these VHmolecules may be better expressed and show a higher solubility byintroducing a mutation changing the Trp on position 103 into Arg. Thisis a much easier and more straightforward mutation than Val37Phe,Gly44Glu, Leu45Arg and Trp47Gly or part of it as originally carried outby Davies and Riechmann (1994). It has the additional advantage that itcould be performed on all VH sequences, not only of human origin, but ofall other species having antibodies with an Ig-fold.

Example 2 Generation of Soluble Single Domain from a scFv AntigenBinding Fragment

In cases where a minimal size of the antigen binding fragment isenvisaged, it might be an advantage to design a single domain from anexisting scFv. The VH domain has specific interest since this domain, inprinciple, provides specificity and is the largest contributor toaffinity. Single domains have further advantages due to their smallersize. Although it has been repeatedly shown that VH domains retainsufficient activity to interact with antigen, VH domains are known to besticky and insoluble. The present invention shows that these problemscan be remedied by the Trp103Arg substitution.

Example 3 Effect of Arg103Trp Mutation on Solubility and Antigen Bindingof VHH

3.1 Production and Purification of Wild Type and Mutant VHH

The gene fragment encoding the anti-β-lactamase VHH TEM04 wasmutagenized by PCR using the FR4 specific primer A4short-TEM04 (5′-GGAGAC GGT GAC CTG GGT CCC CTG GCC CCA TAC GAC-3′) (SEQ ID NO: 96) therebychanging the wild type residue Arg on position 103 to Trp103. Using asimilar approach, the anti-carcinoembryonic antigen (CEA) VHH CEA71, wasmutated with primer A4short-CEAVH (5′-GGA GAC GGT GAC CTG GGT CCC CTGGCC CCA GGG GC-3′) (SEQ ID NO: 97). The E. coli production vector pHEN6was used for expression of the wild type and mutated VHH fragments.pHEN6 is derived from pHEN1 (Hoogenboom et al. (1991)), pHEN6 encodingthe hexahistidine tag sequence for purification of VHH and lacking thephage M13 gene 3. The PCR-products and vector were digested withNcol-BstEll and loaded on a 1% agarose gel. Fragments and vector werepurified from gel with Jetsorb, ligated, transformed into WK6 competentcells and plated onto LB agar plates containing 100 μg/ml ampicillin and2% glucose. Mutation of R to W on position 103 was confirmed bysequencing (FIG. 1).

For each construct pre-cultures were started in triplicate in 10 ml ofLB-medium containing 100 μg/ml ampicillin and 2% glucose. 330 mlcultures (in TB-medium with 100 μg/ml ampicillin) were inoculated with 3ml of preculture and grown at 37° C. Cultures were induced at OD600nm=0.4 with 1 mM IPTG and grown overnight at 28° C. No significantdifferences were observed in cell densities after induction between thewild type and mutant (VHH TEM04: OD600(wt)=1.05.+−.0.25;OD600(mut)=1.25.+−.0.05; VHH CEA71: OD600(wt)=1.48.+−.0.28;OD600(mut)=1.30.+−.0.20), suggesting that no toxic products wereexpressed.

The cells were boiled in reducing sample buffer and loaded on 15% PAGE,normalized for the number of cells (OD600 nm=0.1). The proteins wereblotted on nitrocellulose and blocked overnight in PBS containing 1%casein. The hexahistidine-tagged VHH was detected with mouseanti-Histidine monoclonal antibody (Serotec, diluted 1:1000 in PBS) andafter 4 washes with PBS-0.5% tween-20 incubated with anti-mouse alkalinephosphatase conjugate (Sigma, diluted 1:1000) using NBT and BCIP aschromogenic substrates. It can be concluded that the VHH TEM04 mutant isexpressed at much lower levels than its wild type derivative, while forVHH CEA71 no differences were observed (FIG. 2).

Periplasmic extracts were made from all cultures by resuspending thecells in 4 ml TES (0.2 M Tris-HCl, 0.5 mM EDTA, 0.5 M sucrose; pH 8.0).The suspension was incubated for 30 minutes on ice. Subsequently 6 ml0.25×TES was added and the incubation on ice was continued for 20minutes. Periplasts were removed by centrifugation for 20 minutes at10,000 rpm and 4° C. in SS34-rotor (Sorval). VHH was purified by IMACusing Ni-NTA (QIAGEN). The yields were determined by measuring the OD280nm using the calculated molar extinction coefficients (VHH TEM04E(R103)=2.168 and E(W103)=2.582; VHH CEA71 E(R103)=1.444 andE(W103)=1.865) (Table 1). As was observed by Western blot analysis theyield of mutant antibody for VHH TEM04 was much lower than for its wildtype, while for VHH CEA71 no difference was found. The purified VHH wereanalyzed on a coomassie stained PAGE (FIG. 3), which revealed thatβ-lactamase was co-purified as a complex with VHH TEM04, both for thewild type and the mutant form.

TABLE 1 Production yields of wild type (R on position 203) and mutant(W) VHH expressed per liter of culture. Yield (mg/l) VHH R (wild type) W(mutant) TEM04 14 ± 5 2.3 ± 0.7 CEA71 33 ± 4 32 ± 2 3.2 Antigen Binding Characteristics of VHH CEA71 Variants.

An ELISA was performed to compare the antigen binding characteristics ofthe wild type and the mutant VHH CEA71. A microtiter plate (Maxisorp,NUNC) was coated overnight at 4° C. with CEA (Scripps) at aconcentration of 1 μg/ml (in PBS), and blocked for two hours at roomtemperature (RT) with 300 μl 1% casein in PBS. The plates were washedthree times with PBS-tween. Dilution series (10 μg/ml to 4.57 ng/ml,dilution factor three) of all purified samples were incubated intriplicate (100 μl/well) for 2 hours at RT. Plates were washed six timeswith PBS-tween, after which binding of VHH was detected by incubationwith mouse anti-Histidine mAB (Serotec; 1:1000 diluted; 1001/well) for 1hour at RT followed by anti-mouse-alkaline phosphatase conjugate (Sigma,1:2000 diluted), also for 1 hour at RT. Staining was performed with thesubstrate PNPP (p-nitrophenyl-phosphate, 2 mg/ml in 1M diethanolamine, 1mM Mg₂SO₄, pH9.8) and the signals were measured after 30 minutes at 405nm. The CEA wild type VHH still binds at approximately tenfold lowerconcentrations than the mutant form (FIG. 4). This means that either 90%of the mutant protein is not correctly folded (thus inactive) or thatthe affinity of the mutated VHH is tenfold lower. For VHH TEM04 no ELISAwas performed, but on the coomassie stained gel (FIG. 3) the co-purifiedβ-lactamase seems to have a similar intensity as the VHH, suggestingthat the R103W mutant is produced completely in an active form. Ittherefore can be assumed that the introduction of Tryptophan on position103 decreases the affinity.

3.3 Solubility of VHH CEA71 Wild Type and R103W Mutant.

The solubility characteristics were examined by determination of theconcentration of ammonium sulfate, at which the VHH starts toprecipitate. Therefore a saturated stock solution of ammonium sulfatewas prepared by dissolving an excess of salt in a limited volume ofwater. After equilibration for 2 hours at RT, the solid particles wereremoved by centrifugation at 4300 rpm for 10 minutes and the supernatant(100% ammonium sulfate solution) was used to make dilutions of 0-80%. 60μl sample was added to 300 μl ammonium sulphate solution and mixed for18 hours at 4° C. This mixture was centrifuged for 10 minutes at 13000rpm in an Eppendorf centrifuge. The amount of soluble VHH in thesupernatant was determined in ELISA deduced from the degree of antigenbinding (FIG. 5A) and on the other hand by measuring the proteinconcentration with OD280 (FIG. 5B).

From both types of measurements it can be concluded that the wild typeanti-CEA VHH CEA71 started to precipitate at an ammonium sulfateconcentration of 65%, while the mutant form shows signs of precipitationat 58%. This experiment clearly demonstrates that the introduction ofTryptophan on position 103 in the context of the wild type sequence ofVHH CEA71, which has Arginine on this position, decreases itssolubility.

Example 4 Cloning, Selection and Production of Functional Single DomainHeavy Chain Antibody Fragments

Dromedaries and llamas were immunized intramuscularly with a cocktail ofantigens using Freund's complete adjuvant (first injection) and Freund'sincomplete adjuvant (subsequent injections). Dromedaries were immunizedwith CEA (Carcino Embryonic Antigen), ovalbumine (OVA), PSA (ProstateSpecific Antigen), Variant Surface Glycoprotein trypanosome (VSG),β-lactamase, carbonic anhydrase, Cutinase, Potyvirus and Lysozyme.Llamas were immunized with Poly A Binding Protein Type 2 (PABP2), LinoicAcid BSA-conjugate, a humanized mouse mAb to CD40 (Hu-anti-CD40), humanserum albumin (HSA), Salmonella typhimurium, Rotavirus. Following 6injections with a one-week interval, a blood sample of 100 ml wascollected. PBL cells were separated on a Ficoll-Paque Plus gradient(Amersham Biosciences). Total RNA was isolated from these cells using anacid guanidinium thiocyanate extraction (Chomczynski and Sacchi, 1987)and cDNA was prepared using M-MLV RT (Gibco BRL) and randomoligonucleotide primers (Amersham Biosciences). With the primers Call001(5′-TCCTGGCTGCTCTTCTACAAG-3′) (SEQ ID NO: 98) and Call002(5′-GGTACGTGCTGTTGAACTGTTCC-3′) (SEQ ID NO: 99), annealing to the leadersequence and the CH2 exon of all camelid immunoglobulins respectively,the gene fragments coding for the variable domain were amplified by PCR.To introduce a Ncol restriction site, the gene fragments werereamplified using an equimolar mixture of upstream primers SM017(5′-CAGCCGGCCATGGCTGATGTGCAGCTGGTGGAGTCTGG-3′ (SEQ ID NO: 101), andSM018 (5′-CCAGCCGGCCATGGCTCAGGTGCAGCTGGTGGAGTCTGG-3′) (SEQ ID NO: 100)in combination with Cll002 in a nested PCR. In the final amplificationthe A4short primer (5′-CATGCCATGACTCGCGGCCCAGCCGGCCATGGC-3′) (SEQ ID NO:102) was used on one hand to introduce a Sfil site and on the other handthe primers as described before were used to introduce the key residueson position 103 and a Notl site: primer 1 (R103), primer 2 (R103),primer 3 (K103), primer 4 (Q103), primer 5 (L103), primer 6 (F103)primer 7 (G103), primer 8 (S103), primer 9 (P103), primer 10 (Y103). ThePCR products were cloned using the Sfil/Notl restriction enzymes intothe phagemid vector pHEN4 (a derivative of pHEN1 (Hoogenboom et al.,1991) with a HA-tag downstream the cloning sites of the heavy chainantibody fragment). The repertoire was expressed on phage followinginfection with M13K07 helper phages. Specific binders were selectedusing the principle of phage display and panning (Ghahroudi et al.,1997).

Single domain heavy chain antibodies specific for CEA (CEA1 and CEA72),PSA (N3-A, N8-B, C9-B, C11-B, C12-A, C1-B, C24-A, N13-A, N15-B), HSA(ALB1, ALB2, ALB3, ALB4, ALB5), Hu anti-CD40 (CD40-1, CD40-2, CD40-3,CD40-4, CD40-5, CD40-6, CD40-7), OVA (B13, 1DBOVA11, 1DBOVA23, 1DBOVA43,A2-19, A4-17, B368, R24), VSG (cAbAnO4), β-lactamase (cAbBLA01,cAbTEM04), carbonic anhydrase (1 D2CA30), PABP2 (C4PABP2, E3 PABP2, F6PABP2), Salmonella typhimurium (MPOD6 salmon), Rotavirus (1-F6 RTV),Linoic Acid (LA-1), Cutinase (CutIII19, A4cut9, CACU13, CABCUT4, CU16),Potyvirus (48dpvy, 348DPVY, 1648DPVY, 1048DPVY23, PVYIA15, PVYIA2,PVYIA1, PVY17) and Lysozyme (1D2L28) were isolated and evaluated forexpression, binding in ELISA and affinities. The sequences are listedbelow; amino acid position 103, as determined by the Kabat numbering isindicated in bold,

N3-A (SEQ ID NO: 103) DVQLQESGGSLVQPGGSLRLSCAASGFTFSAYYMIWVRQAPGKGLEWVSGISANGRDTLYEDSVEGRFAISRDNAKNTLYLQMNSLRSEDTALYYCVIGA LITGRRGQGTQVTVSS N8-B(SEQ ID NO: 104) DVQLQESGGGLVQPGGSLRLSCAASGFLFSDTYMTWARQAPGKGLEWLGGISKDGSGTLYEDSVEGRFTISRDNAKNTLYLQMNSLKSEDTALYYCSTGA LLPTRPQGQGTQVTVSSC9-B (SEQ ID NO: 105) DVQLQESGGGLVQPGGSLRLSCAASGFTFSNHYMTWVRQAPGKGLEWVSVISNDGRYTDYADSVKGRFTISRDNAKNTLYLQMNSLKTEDTALYTCVRGY YLTNLPAGDRGQGTQVTVSSC11-B (SEQ ID NO: 106)DVQLQESGGGLVQPGGSLRLSCAASGFIFSNTYMTWVRQAPGKGLEWVSGISADGRDTLYADSVEGRFAISRDNAKNTLYLQMNSLRSEDTALYYCVTGA LMTGRRGQGTQVTVSSC12-A (SEQ ID NO: 107)DVQLQESGGGLVRPGGSLRLSCAASGFLFSGTYMTWARQAPGKGLEWLCGINKDGSGTLYADSVEGRFTCSRDNAKNTLYLQMNSLKSEDTALYYCSTGA LLPTRPQGQGTQVTVSSC1-B (SEQ ID NO: 108) DVQLQESGGGLVQPGGSLRLSCAASGFTFSTSYMTWARQAPGKGLEWVSGINRDGNNPLYADSVEGRFTVSRDNAKNTLYLQMNSLKSEDTALYYCVAGA LVAGARGQGTQVTVSSC24-A (SEQ ID NO: 109)DVQLQESGGGLVQPGGSLRLSCAASGFAFTPSYMSWVRQAPGKGLEWVSVISNDGRYTDYADSVKGRFTISRDNAKNKTLYLQMNSLKTEDTALYTCVRG YYLTNLPAGDRGQGTQVTVSSN13-A (SEQ ID NO: 110)DVQLQESGGGSVQPGGSLRLSCAASGFTFKDASMNWVRQAPGKGLEWVSAINGGGTVTDYADPMEGRFTISRDNA KNTLYLQMNSLNFEDTALYYCATG WLFRANNYRGQGTQVTVSSN15-B (SEQ ID NO: 111)DVQLQESGGGSVQAGGSLRLACAATAYTYDSNVLGWFRQAPGKEHEGVAVIYTGTRTTYYADSVKGRFTISQDNAKNTVYLQMNSLKPGDTAMYFCAANV RLGGVWSFDYRGQGTQVTVSSALB-1 (SEQ ID NO: 112)QVQLQESGGGLVQPGGSLRLSCAASGFAFSSFPMTWVRQAPGKGLEWVSGILEGGGSPAYADSVKGRFTISRDDAKNTLYLQMNSLKPEDTAVYYCAKGY VYAREGARSQGTQVTVSSALB-2 (SEQ ID NO: 113)QVQLQESGGGLVQPGGSLRLTCTASGFAFSNFGMSWVRQPPGKGLEWVSAISADSSTKNYADSVKGRFTISRDNTKKMLYLEMNSLKPEDTAVYHCVIGR GSASSQGTQVTVSS ALB-3(SEQ ID NO: 114) QVQLQESGGGLVQPGNSLRLSCAASGFAFGNFGMSWVRQAPGKEPEWVSSIDSIGSDTLYADFVKGRFTISRDNAKSTLYLQMNSLKPEDTAVYYCTIGG SLSRSSQGTQVTVSS ALB-4(SEQ ID NO: 115) QVQLQESGGGLVQPGNSLRLSCAASGFSFRSFGMSWVRQAPGKGPEWVSSINSSGDDTRYTDSVKGRFTISRDNAKSTLYLQMNSLKPEDTAVYYCTIGS SISRSSQGTQVTVSS ALB-5(SEQ ID NO: 116) QVQLQESGGGLVQPGGSLRLTCTASGFAFSSFGMSWVRQPPGKGLEWVSAISADSSTKNYADSVKGRFTISRDNDKKMLYLEMNKLKPEDTAVYHCVIGR GSPSSQGTQVTVSS CEA1(SEQ ID NO: 117) QVQLVESGGGLVQPGGSLRLSCAASGFTFSKYDMSWVRQAPGKGLEWVSRISSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATPT YSSDYRGLPPGQGTQVTVSSCEA72 (SEQ ID NO: 118)QVQLVESGGGLVQPGGSLRLSCAASEFTFSSSYMSWVRQAPGKGLEWVSGINTDGSFTRYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTALYYCAVGG GLGYGPRGQGTQVTVSS B13(SEQ ID NO: 119) QVQLQASGGGLVQPGGSLRLSCAASGFDFMNVYMTWVRQAPGKGVEWVSGISVSGSITHYSESVKGRFTISRDNAKNMLYLQMNSLKSEDTARYYCARGG YNRYYGALGQGTLVTVSS1DBOVA11 (SEQ ID NO: 120)QVQLVESGGGSVQ?GESLRLSCVASGFTFDV?YMNWVRQAPGKGLEWVSGISASGY?TTYA??VKGRFTISRDNAKNTLYLQMNSL??TRGQGTQVTVSS 1DBOVA23 (SEQ ID NO:121) QVQLVESGGGSVQAGGSLIISCAASGFDFSNNYMTWVRQAPGKGVEWVSGISVSGSITHYTDSVKGRFTISRDNAKNTLYLQMNSLRSEDTARYYCGTGG YGRYYGTLGQGTQVTVSS1DBOVA43 (SEQ ID NO: 122)QVQLVESGGGLVQPGGSLRLSCVGSGFTFSSYYISWVRQAPGKGLEWVSGISGSGATTSYTDSVKGRFTISRDNAKNTVYLQLNSLETEDSAMYYCRLGY GTPPGGVWPSQRQGTQVTVSSA2-19 (SEQ ID NO: 123)QVQLQASGGGLVQPGGSLKLSCVVSGFLFSNYAFSWVRQAPGKGLEWVSTIGTSSGYTNYAPSVKGRFITSRDNAKNTVYLQLNSLKTEDTAMYYCRRPG TDERGQGTQVTVSS A4-17(SEQ ID NO: 124) QVQLQASGGGLVQPGGSLRLSCAASGFDFSNVYMTWVRQAPGKGVEWVSGISVSGSITHYSDSVKDRFTISRDNAKNTLYLQMNSLKSEDTARYYCARGG YNTYSGALGQGTQVTVSSB368 (SEQ ID NO: 125) VQLVESGGGSVQAGGSLILSCTASGLPYKSYCMGWFRQAAGKEPEGVATINSGTGSKFYTDSVKGRFTISLDNDNNRVYLEMSSLKPEDTATYYCAAGQRHSCGYVLKNTDGWTHRAQGTQVTVSS R24 (SEQ ID NO: 126)SAQVQLQASGGGLVQPGGSLKLSCVVSGFLFSNYAFSWVRQAPGKGLEWVSTIGTSSGYTNYAPSVKGRFTISRDNAKNTVYLQLNSLKTEDTAMYYCRR PGTDERGQGTQVTVSScAbAn04 (SEQ ID NO: 127)QVQLVESGGGSVEAGGSLRLSCVVSGYSVSIGCMAWFRQAPGSGREGVAGISRGGSMTDYTASVKGRFTISRD-ND QRTVTLQMNSLKPEDTAVYYCARDGPEIATMIGGSRGRGTQVTVSS cAbBLA01 (SEQ ID NO: 128)QLQLVESGGGSVQSGGSLRLSCKVSGYIGSTNCMGWFRQAPGKEREGVASLFTGSGNTYYGDSVKGRFTISEDNAKNTVSLQMNSLKPEDTAMYYCASSSNVGSDESCGRKNTRQFVYTYQGQGTQVTVSS cAbTEM04 (SEQ ID NO: 129)QVQLVESGGGLVQAGGSLRLSCAASGFTFSSAWMTWVRQAPGKGLEWVTSIATDGSTDYADSVKGRFTISRDNAKNTLYLQLNSLNTEDTAVYYCAKDRW GYVVRGQGTQVTVSS1D2CA30 (SEQ ID NO: 130)QVQLVESGGGSVQAGGSLRLSCAASGYTVSTYCMGWFRQAPGKEREGVATILGGSTYYGDSVKGRFTISQDNAKNTVYLQMNSLKPEDTAIYYCAGSTVASTGWCSRLRPYDYHYRGQGTQVTVSS C4 PABP2 (SEQ ID NO: 131)QVQLQESGGGLVQPGGSLRLSCAASGFTFSRSWMYWVRQAPGKGLEWVSSITPGGSEPFYVDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYFCAKDS KNGPRGQGTQVTVSS E3PABP2 (SEQ ID NO: 132)QVQLQESGGGLVQPGGSLRLSCAASGFTFSRSWMYWVRQAPGKGLEWVSSITPGGTEAFYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTALYFCAKDS KNGPRGQGTQVTVSS F6PABP2 (SEQ ID NO: 133)QVQLQESGGGLVQPGGSLRLSCATSGFISDYWMYWVRQAPGKGLEWVSSITPGASTTLYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKGSK IGPRGQGTQVTVSS LA-1(SEQ ID NO: 134) QVQLQDDSGGGLVQPGGSLKLSCAASGFTFSNYEMSWVRQAPGKGLEWVSSINNGGDITYYANSVKGRFTISRDNTKNTLYLQMNSLKSEDTAVYYCKVP NRRLRGPGTQVTVSSCD40-1 (SEQ ID NO: 135)QVQLLVESGGGLVQPGGSLRLSCAAAGFTFSNYAMSWVRQAPGKGLEWVSGIKSGGGRTYYADSVKGRFTISRDNAKNTLTLQLNSLKTEDTAMYYCAKGARYDSDYDVYTWLDSYSGQGTQVTVSS CD40-2 (SEQ ID NO: 136)EVQLVESGGGLVQAGGSLELSCSFGGRAFDRYFMAWFRQAPGKGLEWVSRIYSGGSTSYADSVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCDIAGR RGQGIQVTVSS CD40-3(SEQ ID NO: 137) EVQLVESGGGLVQAGDSLRLSCAASGRTFNTVDMGWFRQAPGKEREFVAHISWRGGSTYYADSVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCDIAG RRGQGTQVTVSS CD40-4(SEQ ID NO: 138) QVQLVESGGGLVQPGGSLRLSCAASGFAFSRYSMYWVRQAPGKGLEWVSEIYPDGNGWYTSSVKGRFTISRDNDKNMLYLQMNSLKPDDTAVYYCALSRS GQGRGQGTRVTVSS CD40-5(SEQ ID NO: 139) EVQLVESGGGLVQAGGSLELSCSFGGRAFDRYFMAWFRQAPGKGLEWVSRIYSGGSTSYADSVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCDIAGR RGQGIQVTVVS CD40-6(SEQ ID NO: 140) EVQLVESGGGLVQAGDSLRLSCAASGRTFNTVDMGWFRQAPGKEREFVAHISWRGGSTYYADSVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCDIAG RRGQGTQVTVSS CD40-7(SEQ ID NO: 141) AVQLEESGGDSVQAGGSLRLSCAASGFTFSRYSMYWVRQPPGKGLEWVSEIYPDGNGWYTSSVKGRFTISRDNDKNMLYLQMNSLKPDDTAVYYCALSRS GQGRGQGTRVTVSS MPOD6Salmon (SEQ ID NO: 142)QVQLQESGGGLVQPGGSLRLSCAASGFTFNDYFMNWVRQAPGKGLEWVSGIYSDGSKTYYGDSVKGRFTISRDNAKNTLYLQMNSLKSEDSAVYYCTRGT GWSSTPYTYRGQGTQVTVSS1-F6 RTV (SEQ ID NO: 143)QVQLQEVRGRLVQLGGSLRLSCAASGFTFKYYAMSWVRQAPGKGLEWVSYINDNGGYTDYSDSVKGRFTISRDNAKNTLYLHMNRLKPEDTAVYFCAKWDTDAVSSSRYKTHNGDIRGPGTQVTVSS CUTIII19 (SEQ ID NO: 144)QVQLVESGGGLVQAGESLTLSCTASGGSFNNWHMGWFRQAPGTEREFVAAIRRAYGSTFYADSVKGRFTIARDNAKNTVYLQMSSLKPEDSAVYYCAAKR AFRVGGDFEYYGQGTQVTVSSA4cut9 (SEQ ID NO: 145)QVQLQASGGGLVQPGGSLRLSCAASGFTFSTYYMNWVRQAPGKGLEWVPGINKDGSVSHYADSVKGRFTISRDNAKNTLYLRMNSLKSEDTALYYCATIA GFRVGGGPGGTQVTVSSCACU13 (SEQ ID NO: 146)DVQLVESGGGLVQPGGSLRLSCAASGFRFDSVAMTWVRQTPGKGLEWVSSISWDGTTTSYAASVKGRFTISRDNAKNTLYLQLDSLKTEDTAMYYCTKTG VDYRDSRDRGRGTQVTVSSCABCUT4 (SEQ ID NO: 147)QVQLVESGGGLVQPGGSLRLSCAASGFRFDSVAMTWVRQAPGKGLEWVSSISWDGTTTSYAASVKGRFTISRDNAKNTLYLQLDSLNTEDTAMYYCTKTG VDYRDSRSRGQGTQVTVSSCu16 (SEQ ID NO: 148) QVQLVESGGGSVQAGGSLKLTCELSGFNGRSNCMGWFRQVLGKDREGVAAINHPEGSEFYDDSVKGRFKITRDGLKDADSLQMNNLKPEDTATYYCALRPYDCYSGAWSPADFYYRGARGTQVTVSS 48dpvy3 (SEQ ID NO: 149)QVQLQASGGGSVEAGGSLRLSCAASGDTAKLNCMAWFRQAPGKERERVASLSTRLTTTSYTDSVKGRFTISQDTATNTVYLEMNSLQPEDTAVYYCQLSR GGTNYRGQGTLVTVSS48DPVY16 (SEQ ID NO: 150)QVQLQASGGGSVQAGGSLRLSCAASGYTYSSNCMGWFRQALGKEREGVAAIYTGGGSTYYADSVKGRFTISQDNAKNTVLYQMNSLKPEDTAMYYCAASLLPLVAGIGVWDAFDYRGQGTQVTVSS 48DPVY10 (SEQ ID NO: 151)QVQLQASGGGSVQAGGSLRLSCVASQYEYSNNYIAWFRQAPGKEREGVAAIYTGGVTRASPYYADPVKGRFSISKDNAKNTVYLQMNDLKPEDSGTYICASSIHGLGNPLRSEFSYYGQGTLVTVSS 48DPVY23 (SEQ ID NO: 152)QVQLQASGGGSVEAGGSLRLSCAASGDTAKLNCMAWFRQAPGKERERVALLSTRLTTTSYTDSVKGRFTISQDTATNTVYLEMNSLQPEDTAIYYCAARWAGRSCLVSVYDYYGQGTLVTVSS PVYIA15 (SEQ ID NO: 153)QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSGIKSGGGRTYYADSVKGRFTISRDNAKNTLTLQLNSLKTEDTAMYYCAKGARYDSDYDVTWLDSYSGQGTQVTVSS PVYIA2 (SEQ ID NO: 154)DVQLVESGGGSVQAGGSLRLSCTASGLRLNTYHMSWVRQAPGKGLEWVSTIYIGGTTTSHANSVSGRFTISRDDAKNTLYLQMNNLKPEDTAVYFCATGS VNAYGVKGQGTQVTVSSPVYIA1 (SEQ ID NO: 155)QVQLVESGGGSVQAGGSLTLSCTVSGYDFNRCSMNWYRENPGKEREFVAGIDSDGTTTYADSVKGRFTISHDNTRNTLYLQMNSLKSEDTALYYCRLGGL RTWPQYGYRGQGTQVTVSSPVY17 (SEQ ID NO: 156)QVQLVESGGGLVQPGGSLRLSCTASGLRLNTYHMSWVRQAPGKGLEWVSTIYIGGTTTSHANSVSGRFTISRDDAKNTLYLQMNNLKPEDTAVYFCATGS VNAYGVKGQGTLVTVSSAA1D2L28 (SEQ ID NO: 157)QVQLVESGGGLVQPGGSLRLSCAASGFAFSIYRMSWVRQAPGQGLEWVSSIDSGGGITYYADSVKGRFTISRDNAKNTLYLQLNSLKTEDTAMYYCARGHYLYDDIFTGAKGQGTQVTVSSGR

For each construct 10 ml pre-cultures were started in TB containing 100μg/ml ampicillin and 2% glucose. For each pre-culture, 4×330 ml culturewas started at 37° C. in TB containing 100 μg/ml ampicillin with 3 ml ofthe overnight culture. Cultures were induced with 1 mM IPTG at OD600nm=0.4 and grown overnight at 28° C.

Periplasmic extract was prepared for all overnight cultures. Theovernight cultures were centrifuged for 10 minutes at 10,000 rpm at 40°C. The supernatant was removed and the pellet was re-suspended in 16 mlTES (0.2 M Tris-HCl, pH=8.0, 0.5 mM EDTA and 0.5 M sucrose). Thismixture was incubated for 30 minutes on ice. 24 ml 0.25×TES was added,incubated on ice for 20 minutes and centrifuged for 20 minutes at 10,000rpm. The supernatant was purified on Ni-NTA (QIAGEN), and dialyzedovernight against PBS. OD280 was measured and the yield (in mg) ofpurified material per liter of culture was determined. Kd's weredetermined on BIAcore are given in the table below.

Expression level (mg/1 culture, Antigen recognized Kd purified Name ofbinder by antibody (nM) material) Host N3-A Prostate specific — 0.25Dromedary antigen N8-B Prostate specific 1.6 6.2 Dromedary antigen C9-BProstate specific 3.9 2.1 Dromedary antigen C11-B Prostate specific 2.66.1 Dromedary antigen C12-A Prostate specific 2.6 1.2 Dromedary antigenC1-B Prostate specific 0.8 0.75 Dromedary antigen C24-A Prostatespecific — 5.0 Dromedary antigen N13-A Prostate specific — 0.25Dromedary antigen N15-B Prostate specific — 0.65 Dromedary antigen CEA1Carcino Embryonic Dromedary Antigen CEA72 Carcino Embryonic 8.4Dromedary Antigen B13 ovalbumin <13 Dromedary 1DBOVA11 ovalbuminDromedary 1DBOVA23 ovalbumin Dromedary 1DBOVA43 ovalbumin DromedaryA2-19 ovalbumin Dromedary A4-17 ovalbumin Dromedary B368 ovalbuminDromedary R24 ovalbumin Dromedary cAbAn04 Variant surface Dromedaryglycoprotein trypanosome cAbBLA01 β-lactamase <1 0.4 Dromedary cAbTEM04TEM1 Dromedary 1D2CA30 Carbonic anhydrase Dromedary A4Cut9 CutinaseDromedary CACU13 Cutinase Dromedary CABCUT4 Cutinase Dromedary CU16Cutinase Dromedary 48dpvy Potyvirus Dromedary 348DPVY PotyvirusDromedary 1648DPVY Potyvirus Dromedary 1048DPVY23 Potyvirus DromedaryPVYIA15 Potyvirus Dromedary PVYIA2 Potyvirus Dromedary PVYIA1 PotyvirusDromedary PVY17 Potyvirus Dromedary 1D2L28 Lysozyme Llama LA-1 Linoicacid Llama C4 PABP2 Poly A Binding Llama Protein Type 2 E3 PABP2 Poly ABinding Llama Protein Type 2 F6 PABP2 Poly A Binding Llama Protein Type2 CD40-1 humanised mouse 2 8 Llama mAb to CD40 CD40-2 humanised mouseLlama mAb to CD40 CD40-3 humanised mouse Llama mAb to CD40 CD40-4humanised mouse Llama MAb to CD40 CD40-5 humanised mouse Llama mAb toCD40 CD40-6 humanised mouse Llama mAb to CD40 CD40-7 humanised mouseLlama mAb to CD40 MPOD6 Salmonella Llama salmon Typhimurium 1-F6 RTVRotavirus Llama CutIII19 Cutinase Llama ALB-1 Human serum 15 Llamaalbumin ALB-2 Human serum 15 Llama albumin ALB-3 Human serum 15 Llamaalbumin ALB-4 Human serum 15 Llama albumin ALB-5 Human serum 15 Llamaalbumin

Alignment of the CEA1 binder (SEQ ID NO: 117) and a human VH3 germline(DP-47) (SEQ ID NO: 158) revealed a high degree of homology (two aminoacid changes in FR1 on position 1 and 5 and four changes in FR3 onposition 74, 83, 84 and 94), as shown below:

DP-47 EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVSAISGSGGSTYYCEA1 QVQLVESGGGLVQPGGSLRLSCAASGFTFS KYDMS WVRQAPGKGLEWVSRISSGGGSTYYDP-47 ADSVKG   RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK  ----------------------CEA1 ADSVKG   RFTISRDNAKNTLYLQNNSLKPEDTAVYYCAT  PTYSSDYRGLPPGQGTQVTVSS

A specific binder for the CEA tumor antigen, with high homology to thehuman germline gene DP-47 was therefore an ideal candidate to furtherhumanize and evaluate the influence of mutagenesis on binding affinityin ELISA and BIAcore.

Mutagenesis of the residues in FR1 had no significant influence onspecificity, binding affinity and expression level. Mutagenesis of mostof the FR3 residues did not result in loss of specificity, affinity andexpression levels. The most humanized and best performing CEA1 mutantwas chosen for immunogenicity studies. Baboons were immunizedintravenously, intramuscularly and subcutaneously with a weekly dose of1 mg/kg body weight. Blood samples were taken and humoral response wasevaluated in ELISA. No significant antibody response was raised againstthe CEA1 mutant.

Example 5 Construction of a Functional Single Domain Heavy ChainAntibody Fragment Library Derived from Human and Mouse VH

cDNA templates were made starting from blood samples from 20non-immunized human donors (or from other species containing antigenbinding molecules with an Ig-fold). The peripheral blood lymphocytes(PBL) were isolated on Ficoll-Paque gradients (Amersham Biosciences).Total RNA was prepared individually from the 20 samples of PBL's asdescribed in example 4. First strand cDNA was also individuallysynthesized from total RNA with random hexamers as primers (as inexample 4).

Mutagenesis was carried out using a Framework 1 specific primer that is5′ linked to a SfiI site, as backward primer, and the mutagenic primer:

3′-ACC CGA GGA GCC TGG GAC CAG TGG CAG-5′ (SEQ ID NO: 159)

or

3′-ACC CCG GTG AGG AGG GTC CAG TGG CAG-5 (SEQ ID NO: 160)

as forward primer. The DNA obtained was cut with BseRI and ligated tothe double stranded primer:

5′-pC GTC AGG GGC CAA GGA ACC CAG GTC ACC GTC TCC TCA-3′ (SEQ ID NO:161)

3′-CTG CAG TCC CCG GTC CCC TGG GTC CAG TGG CAG AGG AGT-5′ (SEQ ID NO:162) for the PCR product obtained with the first mutagenic primer, or

5′-pAGG GGC CAA GGA ACC CAG GTC ACC GTC TCC TCA-3′ (SEQ ID NO: 163)

3′-GT TCC CCG GTC CCC TGG GTC CAG TGG CAG AGG AGT-5′ (SEQ ID NO: 164)

for the PCR product obtained with the second mutagenic primer.

This primer restores the CDR3 and Framework 4 coding regions. Theresulting DNA was amplified using the same forward primer as above, andusing

3′-C TGG GTC CAG TGG CAG AGG AGT CGCCGGCG-5′ (SEQ ID NO: 165)

as backward primer. This primer created a Notl site at the end of the VHcoding sequence. The resulting material was cut with Sfil and Notl andthe resulting fragment cloned in pHEN4 to yield a phage display library.

The library was selected with a panel of antigens, such as Human SerumAlbumin and human IgG1, thereby obtaining large numbers of antigenspecific antibody fragments. Sequence analysis revealed the introducedresidue on position 103. The produced and His6-tagged VH fragmentsshowed good solubility characteristics, good specificity (not reactiveagainst irrelevant antigens) and high affinities (range of 2 to 50 nM).

A mouse was immunized with a set of antigens (CEA and PSA) and afterfour weeks the spleen was removed, homogenized in guanidiniumthiocyanate buffer with a Polytron homogenizer, debris removed by lowspeed centrifugation and total RNA extracted using the method describedbefore. As was described above the mouse VH encoding gene segments wereamplified thereby introducing the variant residues on position 103. Uponselection high affinity VH fragments were selected with goodcharacteristics with respect to solubility.

Example 6 Isolation, Sequencing and Production of Human TNF SpecificFragments

Selection of binders for tumor necrosis factor α (TNF) from a phagelibrary was carried out as described in Example 4. After panning thelibrary, 48 individual clones were selected and tested in a phage ELISAon immobilized TNF and BSA.

The clones for which the signal on TNF was superior to the one obtainedon BSA were selected for further characterization. By sequencing theselected clones, we were able to eliminate identical clones and todemonstrate that W103 was no longer encoded in the selected fragments.

The gene segments encoding the selected antibody fragment were reclonedin the pHEN6 expression vector as described in example 3, which allowedus to produce the recombinant antibody fragment as soluble periplasmicproteins. The recombinant antibody fragments were purified tohomogeneity from the periplasmic fraction by IMAC on Ni-NTA agarose andsubsequent gel-filtration chromatography on Superdex-75. The purity wasdetermined by SDS-PAGE.

6.1 Applications of anti-TNF Antibody Fragments.

6.1.1 Therapeutic Applications.

Anti-TNF specific fragments were tested in L929 murine fibrosarcomacells following the protocol as described by Ameloot et al. (2001). L929cells were seeded in 96-wells microtiter plates at 30,000 cells perwells. The next day, purified recombinant antibody fragments were addedto some wells, whereas only PBS was added to control wells. In thisexperiment the final concentration of antibody fragment was 1micromolar.

Subsequently a lethal dose of TNF was added to those wells whereantibody was added and also to part of the control wells. After 18hours, the level of surviving cells was estimated by the calorimetricmethod. In this way we demonstrated that these antibody fragments havethe ability to neutralize the cytotoxic effect of TNF and havetherapeutic potential.

6.1.2 Diagnostic Application of Anti-TNF VHH in ELISA.

To individual wells of a microtiter plate, we added 100 μl of theantibody fragments at a concentration of 5 μg/ml in PBS. Afterincubation overnight at 4° C. the plate is blocked with 1% BSA in PBS.The presence of functional immobilized antibody fragment wasdemonstrated by the binding of biotinylated TNF at 1 μg/ml. The presenceof bound biotinylated TNF was demonstrated with streptavidin-alkalinephosphatase conjugate and subsequent reaction withpara-nitrophenyl-phosphate.

6.1.3 Application of Anti-TNF VHH in Antibody Arrays.

Two different formats of antibodies arrays were tested, i.e. onnitrocellulose filters and glass slides. For the filter method 2 μl ofthe purified antibody fragments at concentration of 1 mg/ml in PBS werespotted with a micropipette on a nitrocellulose sheet. After drying, thesheet was blocked with 1% BSA in PBS. The presence of functionalimmobilized antibody fragment on the nitrocellulose sheet wasdemonstrated with biotinylated TNF. The presence of bound TNF wasdemonstrated with streptavidin-alkaline phosphatase conjugate andNBT-BCIP reagent. The appearance of dark spots on those positions wherethe TNF-specific fragments were applied proved that these antibodyfragments retain functionality when passively coated on a solid support.This approach can be used for random screening and selection ofantigen-specific fragments.

For the glass slide type of antibody array the anti-TNF antibodyfragments were covalently immobilized. Purified antibody fragments werediluted to a concentration of 200 μg/ml in PBS containing 20% glycerol.The samples where transferred to wells of a 384 well microtiter plate.Subsequently an automated contact printer was used to deliver 5nanoliter of the antibody solutions to a commercially available glassslide (Telechem-Superaldehyde).

After application of the samples, the glass slide was incubated for 1hour in a humid chamber, to allow the reaction between the reactivealdehydes present on the glass slide and the lysine groups present onthe antibody fragment surface to proceed.

The slide was subsequently blocked with 1% BSA/PBS, subsequentlyincubated with Cy3-modified TNF (fluorescent label Cy3-AmershamBiosciences) at 1 μg/ml and finally washed with PBS to remove unboundlabeled TNF. After scanning the fluorescence intensity present on thesurface of the slide, we observed an enhanced signal at those positionswhere the TNF specific antibody fragments were applied.

This result demonstrated that these antibody fragments were covalentlyimmobilized with retention of binding capacity.

6.1.4 Affinity Chromatography.

Purified antibody fragments at a concentration of at least 1 mg/ml weredialyzed against 0.1 M sodium bicarbonate and subsequently mixed with 1ml gel suspension (CNBr-activated Sepharose) following the protocol asdescribed by the manufacturer (Amersham Biosciences).

After incubation for 3 hr, 100 μl of a 1M Tris pH 8 solution was added.After extensive washing, in order to remove unbound protein, theaffinity resin was resuspended in 1 ml PBS.

The functionality of the immobilized anti-TNF antibody resin was testedas described below. To 1 ml of human plasma 10 μg of purified TNF wasadded. We then added 100 μl of the affinity resin. After of overnightincubation of this suspension, the resin was washed extensively withPBS. The pelleted beads were subsequently resuspended in 100 μl of asolution containing 1% SDS solution and boiled for 10 minutes. Aftercentrifugation 20 μl of the supernatant was loaded on SDS-PAGE. A bandof the expected molecular weight was enriched in the analyzed sample.

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The invention claimed is:
 1. An isolated functional immunoglobulinV-domain from the H-chain of a functional heavy chain antibody (V_(HH))or from the H-chain of a four-chain antibody (V_(H)), wherein the aminoacid at position 45 is leucine (L) or wherein the amino acid at position45 is replaced with leucine (L) and wherein the amino acid at position103 is arginine (R) or wherein the amino acid at position 103 isreplaced with arginine (R), wherein said immunoglobulin V-domain is notstabilized by a disulfide bridge formed between CDR3 and either CDR1 orCDR2, further comprising leucine at position 108, said positionsdetermined according to the Kabat numbering.
 2. An isolated functionalimmunoglobulin V-domain from the H-chain of a functional heavy chainantibody (VHH) or from the H-chain of a four-chain antibody (VH),wherein the amino acid at position 45 is leucine (L) or wherein theamino acid at position 45 is replaced with leucine (L) and wherein theamino acid at position 103 is arginine (R) or wherein the amino acid atposition 103 is replaced with arginine (R), wherein said immunoglobulinV-domain is not stabilized by a disulfide bridge formed between CDR3 andeither CDR1 or CDR2, wherein said immunoglobulin V-domain does not havea cysteine residue in the CDR3 and in either CDR1 or CDR2, and furthercomprising leucine at position 108, said positions determined accordingto the Kabat numbering.
 3. The isolated functional immunoglobulinV-domain of claim 1, further comprising valine (V) at position 37,glycine (G) at position 44 and/or tryptophan (W) at position 47, saidpositions determined according to the Kabat numbering.
 4. The isolatedfunctional immunoglobulin V-domain of claim 2, further comprising valine(V) at position 37, glycine (G) at position 44 and/or tryptophan (W) atposition 47, said positions determined according to the Kabat numbering.5. An isolated polypeptide comprising the isolated functionalimmunoglobulin V-domain of claim 3, wherein said polypeptide is a dimer,trimer or multimer.
 6. An isolated fusion polypeptide comprising theisolated functional immunoglobulin V-domain of claim 4, wherein saidpolypeptide is a dimer, trimer or multimer.
 7. An isolated fusionpolypeptide comprising the isolated functional immunoglobulin V-domainof claim 1, wherein said polypeptide is a dimer, trimer or multimer. 8.An isolated fusion polypeptide comprising the isolated functionalimmunoglobulin V-domain of claim 2, wherein said polypeptide is a dimer,trimer or multimer.
 9. A pharmaceutical composition comprising theisolated functional immunoglobulin V-domain according to any of claims 1to 4, optionally in combination with a suitable excipient.
 10. Apharmaceutical composition comprising the isolated polypeptide accordingto any of claims 5 to 8, optionally in combination with a suitableexcipient.